Antigens associated with endometriosis, psoriatic arthritis and psoriasis

ABSTRACT

Specific binding members that bind the ED-A isoform of fibronectin for use in methods of diagnosis, detection, imaging and/or treatment of endometriosis, and/or for use in delivery to the neovasculature of endometriotic tissue of a molecule conjugated to the specific binding member. Specific binding members that bind tenascin-C, especially the A1, A2, A3, A4 and/or D domain tenascin-C large isoform, for use in methods of diagnosis, detection, imaging and/or treatment of endometriosis, psoriatic arthritis or psoriasis, and/or for use in delivery to the neovasculature of endometriotic, psoriatic arthritic or psoriatic tissue of a molecule conjugated to the specific binding member.

The present invention relates to the detection and treatment ofendometriosis, psoriatic arthritis and psoriasis. The invention involvesuse of a specific binding member that binds the ED-A isoform offibronectin, especially a specific binding member that binds domain ED-Aof fibronectin, or a specific binding member that binds tenascin-C,especially the A1, A2, A3, A4, B and/or D domain of tenascin C (the“long form” of tenascin-C).

Most conventional pharmaceuticals currently in use for the treatment ofangiogenesis-related diseases (such as cancer, arthritis, etc.) do notselectively accumulate at the site of disease [Bosslet et al., 58,1195-1201 Cancer Res. (1998)]. For example, intravenously administereddrugs distribute evenly within the different organs and tissues of thebody, rather than selectively accumulating at the site of disease.

One approach to circumvent the disadvantages of conventionalpharmacological therapies involves the preferential delivery of abioactive agent to the tumor site by means of a binding moleculespecific for a pathology-associated marker [Neri & Bicknell (2005)Nature Rev. Cancer]. The selective targeting of the drug to the diseasedtissue will ultimately result in an increased local concentration at itssite of action, sparing normal organs from the toxic effects of thebioactive agent used to confer a pharmacological benefit (e.g., acytotoxic drug, a cytokine, a radionuclide, a photosensitizer). In mostcases, this will lead to an improved therapeutic index of the deliveredpharmaceutical, i.e. a higher efficacy with minimized side effects.Indeed, the favourable toxicity profile of site-specific therapeuticsmay open new avenues in the therapy of angiogenesis-related diseases,allowing the systemic administration of highly potent and promisingagents, which are currently either given at suboptimal doses or whoseclinical application has to date been impeded by unacceptable toxicitieswhen applied in an unmodified form.

Ligand-based pharmacodelivery strategies fundamentally rely on theidentification of good-quality markers of pathology, allowing aclear-cut discrimination between diseased tissues and healthy organs.Monoclonal antibodies and their fragments represent the preferred agentsfor pharmacodelivery applications [Rybak et al. 2, 22-40 Chem. Med. Chem(2007); Shrama et al., 5, 147-159 Nat. Rev. Drug Discovery (2006)], eventhough globular protein mutants [Binz and Plückthun, 23, 1257-1268Nature Biotechnology (2005)], peptides [Sergeeva et al., 58, 1622-1654,Adv. Drug. Deliv. Rev. (2006)] and even small organic ligands [Low etal., 41, 120-129, Acc. Chem. Res. (2008)] are increasingly being used.Most efforts in the field of disease targeting have been made usingspecific markers expressed on the surface of diseased cells (e.g., onthe surface of tumour cells in cancer). However, targeting antigens ondiseased cells themselves is a complex task for blood-borne agents,facing a number of physical and kinetic barriers, which may preventefficient pharmacodelivery. These barriers include interstitial pressureat site of disease, relatively long diffusion distances within theinterstitium, heterogeneity of antigens at sites of disease, as well asthe so-called “antigen barrier” [Dennis et al., 67, 254-261, Cancer Res.(2007)]. All these factors significantly impair deep tissue penetration.

Proteins which are expressed around pathological blood vessels at sitesof disease, but which are either absent or present at reduced levels innormal tissue, represent particularly attractive targets for thedevelopment of selective and efficient pharmacodelivery strategies[Rybak et al. (2007) ChemMedChem; Trachsel et al., 9, R9, Arthritis Res.Ther. (2007)]. Vascular targets are often more easily accessible fromthe bloodstream to systemically administered agents, overcoming theproblem of access and allowing an efficient delivery of the compound tothe site of disease. Furthermore, the same vascular targeting agentcould be useful not only for the delivery of therapeutic agents but alsofor molecular imaging applications.

As used herein, the term “vascular targeting” is not used to indicatethe inhibition of the target molecule in a signalling pathway (e.g. theinhibition of VEGF signalling by bevacizumab or the inhibition of theBCR/ABL kinase by imatinib). Instead, the target molecule expressed onthe vasculature at sites of angiogenesis-related diseases is used as aneasily accessible binding site for specific ligands, exploiting thevasculature as a scaffold to achieve a site-specific localization ofeffector molecules at the tumour tissue. This fundamental conceptualdifference is clear from the fact that, while some vascular markermolecules have been shown to be very suitable for ligand-directed tumourtargeting applications, their pathophysiological role is still largelyunknown (for example, the extra-domain B of fibronectin; see below).

The present inventors have previously performed extensive work on theantibody-based targeting of markers of tumor angiogenesis, such as theextra-domain B of fibronectin (ED-B) [Schliemann and Neri, 1776,175-192, Biochim Biophys Acta (2008]. ED-B is virtually undetectable innormal adult tissues, except for the endometrium in the proliferativephase, but becomes over-expressed in conditions involving tissueremodelling, with a prominent peri-vascular pattern of staining. Threederivatives of the human anti-EDB antibody L19 [Pini et al. (1998) J.Biol. Chem.] are currently being investigated in Phase I and Phase IIclinical trials for cancer therapy (the radiolabelled product L19-¹³¹Iand the immunocytokines L19-IL2 and L19-TNF). The ability of L19 toselectively localize at angiogenic sites in non-tumoral diseases hasbeen studied in animal models of ocular neo-vasculature [Birchler et al.17, 984-988, Nature Biotechnology (1999)], rheumatoid arthritis[Trachsel et al., 9, R9, Arthritis Res. Ther. (2007)], psoriasis[Trachsel et al., 127, 881-886, J. Inv. Dermatol. (2007)] andatherosclerosis [Matter et al., 95, 1225-1233 Circulation Res. (2004)].

More recently, the present inventors have studied other alternativelyspliced domains of extracellular matrix components, whose patterns ofexpression are less well characterized and which have so far been usedmainly as vascular targets for tumour targeting applications: the ED-Adomain of fibronectin [Rybak et al., 67, 10948-10957 (2007) Cancer Res.]and the domain A1 of tenascin-C [Brack et al., 12, 3200-3208, (2006)Clin. Cancer Res.]. ED-A is specifically recognized by the humanmonoclonal antibody F8 [Villa et al., 122, 2405-2413 (2008) Int. J.Cancer], while the human monoclonal antibody F16 binds the A1 domain oftenascin-C [Brack et al. (2006) Clin. Cancer Res.].

The pattern of expression of the extra-domains A1, A2, A3, A4, B and Dof tenascin-C are similar, being almost undetectable in normal adulttissues, but strongly up-regulated in a multitude of different tumours[Brack et al. (2006) Clin. Cancer Res.; Pedretti et al. (2008) LungCancer, in press; Berndt et al., 132, 537-546, J Cancer Res Clin Oncol(2006); Balza et al., 261, 175-178, FEBS Lett. (1990)]. The term“tenascin-C large isoform” is often used to indicate the form oftenascin-C containing the extra-domains A1, A2, A3, A4, B and D [Borsiet al., 270, 6243-6245 (1995) J. Biol. Chem.; Borsi et al., 66, 632-635(1996) Int. J. Cancer; Carnemolla et al., 154, 1345-1352 (1999) Am. J.Pathol.]. By contrast, the expression of the extra-domain C oftenascin-C is more restricted, being undetectable in normal adulttissues and being found only in certain tumour types, mainly lung cancerand high-grade astrocytomas [Carnemolla et al. (1999) Am. J. Pathol.].

Thus, antibody-based targeted delivery of bioactive agents to sites ofangiogenesis is an attractive therapeutic strategy for cancer treatment,but is largely unexplored for chronic inflammatory diseases. We havepreviously demonstrated that the ED-B domain of fibronectin, a marker ofangiogenesis, is expressed in psoriatic lesions in patients and in amouse model of psoriasis as well as in arthritic paws in thecollagen-induced mouse model of rheumatoid arthritis. Using bothradioactive and fluorescent techniques, the human monoclonal antibodyL19, specific to EDB, was found to selectively localize at sites ofinflammation in vivo, following intravenous administration. Theseresults suggest a therapeutic potential for the L19-based selectivedelivery of bioactive compounds to sites of inflammation (Trachsel,2007; PCT/EP2007/004044).

It has also previously been shown by in-situ-hybridisation that the ED-Adomain of fibronectin can be present in human arthritic specimens(Berndt et al., 1998; Kriegsmann et al., 2004), and the inventors havepreviously shown the expression of ED-A in rheumatoid arthritis[PCT/EP2008/009070].

We show herein the patterns of expression of ED-A and of tenascin-Clarge in other angiogenesis-related non-tumoural diseases, such asmultiple sclerosis, psoriatic arthritis, psoriasis, inflammatory boweldiseases and endometriosis, using identical concentrations ofbiotinylated versions of the F8 and F16 antibodies in SIP format [Borsiet al., 102, 75-85 (2002) Int. J. Cancer; Villa et al., 122, 2405-2413(2008) Int. J. Cancer; Brack et al., 12, 3200-3208, (2006) Clin. CancerRes.]. These diseases are all associated with angiogenesis and aresocially very relevant. Ligand-based pharmacodelivery may open newdiagnostic and therapeutic opportunities for these diseases.

Multiple sclerosis is an autoimmune disease in which the immune systemattacks the nervous system, resulting in demyelination of neurones(Compston and Coles, 359, 1221-1231, Lancet, 2002). As well asdemyelination, multiple sclerosis is also characterised by inflammation.There is no known cure for multiple sclerosis and many existingmedications can have adverse side effects or be poorly tolerated.

Psoriasis is a disease which affects the skin and joints, usually bycausing red, scaly psoriatic plaques to occur on the skin. Thesepsoriatic plaques are areas of inflammation. When psoriasis causesinflammation of the joints, it is known as psoriatic arthritis. Moreeffective treatments of psoriasis and psoriatic arthritis are requiredand one approach would be the targeted delivery of anti-inflammatorycytokines, such as IL-10 or TGF-β, photosensitisers or cytotoxic drugswith cleavable linkers. Such an approach requires effective targeting ofdrugs to areas of inflammation, and the identification of specifictargets expressed in these regions.

The main forms of inflammatory bowel disease are Crohn's disease andulcerative colitis. Crohn's disease can affect any part of thegastrointestinal tract, whereas ulcerative colitis is restricted to thecolon and rectum (Summers et al., 2003). Depending on its severity,treatment of ulcerative colitis may require immunosuppression to controlits symptoms and treatment usually involves the administration ofanti-inflammatory molecules.

Endometriosis is a common medical condition in women, and ischaracterized by growth beyond or outside the uterus of tissueresembling endometrium, which normally lines the uterus (Rock andMarkham, 340, 1264-1267 (1992) Lancet).

Endometriosis is typically seen during the reproductive years, and ithas been estimated that it occurs in approximately 5% to 10% of women.Its main, but not universal, symptom is pelvic pain in variousmanifestations. Further, endometriosis is common in women withinfertility (Buyalos and Agarwal, 12, 377-381, (2000) Curr Opin ObstetGynecol).

A major symptom of endometriosis is severe recurring pelvic pain. Theamount of pain a woman feels is not necessarily related to the extent orstage (1 through to 4) of endometriosis. Some women will have little orno pain despite having extensive endometriosis affecting large areas orhaving endometriosis with scarring. On the other hand, women may havesevere pain even though they have only a few small areas ofendometriosis (Muse K, 31, 813-822 (1988) Clin Obstet Gynecol).

Typical endometriotic lesions show histological features similar toendometrium, namely stroma and endometrial epithelium and glands thatrespond to hormonal stimuli. Older lesions may display no glands, butresidual hemosiderin deposits. To the eye, lesions appear dark blue orpowder-burn black and vary in size; some other lesions are red, white,or non-pigmented.

Additionally other lesions may be present, notably endometriomas of theovary, scar formation, and peritoneal defects or pockets. As normalappearing peritoneum of infertile women reveals endometriosis on biopsyin 6-13% of cases, some lesions may not be visible to the eye.

A health history and a physical examination can in many patients leadthe physician to suspect endometriosis.

Use of imaging tests may identify larger endometriotic areas, such asnodules or endometriotic cysts. The two most common imaging tests areultrasound and magnetic resonance imaging (MRI). However, normal resultson these tests do not eliminate the possibility of endometriosis, asareas of endometriosis are often too small to be seen by these tests.

The only way to confirm and diagnose endometriosis is by laparoscopy, orother types of surgery. The diagnosis is based on the characteristicappearance of the disease, and is corroborated by a biopsy, ifnecessary. Laparoscopy also allows for surgical treatment ofendometriosis (Brosens I., 15, 229-233 (1997) Semin Reprod Endocrinol).

For this reason, the discovery of good-quality vascular markers ofendometriosis provides new opportunities for imaging endometriosis (e.g.through the ligand-mediated delivery of radionuclides andradioisotopes), and for the pharmacodelivery to endometriotic tissue ofbioactive molecules (such as cytokines, hormones, therapeuticradionuclides, or drugs with cleavable linkers).

We show herein that an anti-tenascin-C antibody, such as the F16antibody disclosed herein, is able to give a stronger staining patternon human endometriotic tissue than the anti-ED-B antibody L19.Similarly, an anti-ED-A antibody, such as the F8 antibody disclosedherein, is also able to give a stronger staining pattern on humanendometriotic tissue than the anti-ED-B antibody L19, although thestaining seen with the F8 antibody is not as intense as that seen withthe F16 antibody.

We also shown herein that an anti-tenascin-C antibody, such as the F16antibody disclosed herein, is able to give a stronger staining patternon human psoriatic arthritic tissue than the anti-ED-B antibody L19, orthe anti-ED-A antibody F8.

However, we show herein that staining of samples from patients withulcerative colitis is virtually negative for the anti-tenascin-Cantibody F16, the anti-ED-A antibody F8 and the anti-ED-B antibody L19,with only a weak positivity being observed with F8 in some specimens.

Analysis of tissue sections from pathological specimens of patients withmultiple sclerosis also revealed only very weak positivity with theanti-tenascin-C antibody F16, the anti-ED-A antibody F8 and theanti-ED-B antibody L19.

In the case of components of the modified subendothelial matrix, in vivotargeting performance of antibody derivatives correlates with abundantantigen expression [Borsi et al., 102, 75-85 (2002) Int. J. Cancer;Demartis et al., 28, 534-539 (2001) Eur. J. Nucl. Med.; Tarli et al.,94, 192-198 (1999) Blood; Viti et al., 59, 347-352, (1999) Cancer Res.].Based on the findings described above, we found that the expression ofED-A and of the tenascin-C “large” isoform is not found in allangiogenesis-related diseases, and that endometriosis appears to beparticularly suited for pharmacodelivery using anti-ED-A antibodymolecules, while endometriosis, psoriatic arthritis and psoriasis appearto be particularly suited for pharmacodelivery using anti-tenascin-Cantibody molecules.

Accordingly, ED-A of fibronectin is indicated as a vascular marker ofendometriosis, while tenascin-C (in particular, the “large” isoform oftenascin-C) is indicated as a vascular marker of endometriosis,psoriatic arthritis and psoriasis.

Specific binding members, such as antibody molecules that bind the ED-Aof fibronectin, represent novel agents which may be used for thetreatment of endometriosis, while specific binding members, such asantibody molecules that bind the “large” isoform of tenascin-C,represent novel agents which may be used for the treatment ofendometriosis, psoriatic arthritis, or psoriasis.

In a first aspect, the invention provides a specific binding member,e.g. an antibody molecule, that binds the Extra Domain-A (ED-A) isoformof fibronectin (A-FN) for use in a method of treatment of endometriosis.The invention also provides the use of a specific binding member, e.g.an antibody molecule, that binds the Extra Domain-A (ED-A) isoform offibronectin for the manufacture of a medicament for treatingendometriosis. The invention also provides a method of treatingendometriosis in a patient, the method comprising administering to apatient a therapeutically effective amount of a medicament comprising aspecific binding member which binds the ED-A isoform of fibronectin.Preferably, the specific binding member binds the ED-A isoform of humanfibronectin.

The specific binding member, e.g. an antibody molecule, for use in thisfirst aspect of the invention, may bind the ED-A of fibronectin.

The specific binding member e.g. an antibody molecule, for use in thisfirst aspect of the invention, may be conjugated to a detectable label,a radioisotope, or a bioactive molecule, such as a cytokine, a hormone,a therapeutic radioisotope or a cytotoxic drug. The specific bindingmember may be conjugated to the bioactive molecule by a cleavablelinker.

In a second aspect, the invention provides a specific binding member,e.g. an antibody molecule, that binds the ED-A isoform of fibronectinfor use in the delivery to the neovasculature of endometriotic tissue ofa molecule conjugated to the specific binding member. The invention alsoprovides the use of a specific binding member, e.g. an antibodymolecule, that binds the ED-A isoform of fibronectin for the manufactureof a medicament for delivery to the neovasculature of endometriotictissue of a molecule conjugated to the specific binding member. Theinvention also provides a method of delivering a molecule to theneovasculature of endometriotic tissue in a human or animal, wherein themolecule is conjugated to a specific binding member which binds the ED-Aisoform of fibronectin to form a conjugate and the method comprisesadministering the conjugate to the human or animal. Preferably, thespecific binding member binds the ED-A isoform of human fibronectin.

The specific binding member, e.g. an antibody molecule, for use in thissecond aspect of the invention, may bind the ED-A of fibronectin.

The specific binding member e.g. an antibody molecule, for use in thissecond aspect of the invention, may be conjugated to a detectable label,a radioisotope, or a bioactive molecule, such as a cytokine, a hormone,a therapeutic radioisotope or a cytotoxic drug. The specific bindingmember may be conjugated to the bioactive molecule by a cleavablelinker.

In a third aspect, the invention provides a specific binding member,e.g. an antibody molecule, that binds the ED-A isoform of fibronectinfor use in a method of diagnosis of endometriosis. The invention alsoprovides use of a specific binding member that binds the ED-A isoform offibronectin for the manufacture of a diagnostic product for diagnosingendometriosis. The invention also provides a method of detecting ordiagnosing endometriosis in a human or animal, wherein the methodcomprises the steps of:

-   -   (a) administering to the human or animal a specific binding        member which binds the ED-A domain of fibronectin, and    -   (b) determining the presence or absence of the specific binding        member in sites of endometriosis of the human or animal body,        -   wherein localisation of the specific binding member to site            of endometriosis indicates the presence of endometriosis.

Preferably, the specific binding member binds the ED-A isoform of humanfibronectin.

The specific binding member, e.g. an antibody molecule, for use in thisthird aspect of the invention, may bind the ED-A of fibronectin.

The specific binding member e.g. an antibody molecule, for use in thisthird aspect of the invention, may be conjugated to a detectable label,or a radioisotope.

In a fourth aspect, the invention provides a specific binding memberthat binds the ED-A isoform of fibronectin for use in a method ofimaging endometriotic tissue. The invention also provides use of aspecific binding member that binds the ED-A isoform of fibronectin forthe manufacture of an imaging agent for imaging endometriotic tissue.The invention also provides a method of detecting or imagingendometriotic tissue in a human or animal, wherein the method comprisesthe steps of:

-   -   (a) administering to the human or animal a specific binding        member which binds the ED-A domain of fibronectin, and    -   (b) detecting the binding of the specific binding member to        endometriotic tissue in the human or animal body.

Preferably, the specific binding member binds the ED-A isoform of humanfibronectin.

The specific binding member, e.g. an antibody molecule, for use in thisfourth aspect of the invention, may bind the ED-A of fibronectin.

The specific binding member e.g. an antibody molecule, for use in thisfourth aspect of the invention, may be conjugated to a detectable label,or a radioisotope.

A specific binding member for use in the invention may be an antibodywhich binds the ED-A isoform of fibronectin and/or the ED-A offibronectin, wherein the antibody comprises one or more complementaritydetermining regions (CDRs) of antibody H1, B2, C5, D5, E5, C8, F8, F1,B7, E8 or G9, or variants thereof. F8, D5 and B7 are disclosed in Villaet al., 122, 2405-2413 (2008) Int. J. Cancer, while H1, B2, C5, E5, C8,F1, E8 and G9 (and also F8, D5 and B7) are disclosed in WO 2008/120101.Preferably, a specific binding member for use in the invention is anantibody which binds the ED-A isoform of fibronectin and/or the ED-A offibronectin, comprising one or more complementarity determining regions(CDRs) of antibody B2, C5, D5, C8, F8, B7 or G9, or variants thereof.Preferably, the specific binding member binds the ED-A isoform of humanfibronectin. Most preferably, a specific binding member for use in theinvention is an antibody which binds the ED-A isoform of fibronectinand/or the ED-A of fibronectin, comprising one or more complementaritydetermining regions (CDRs) of antibody F8, or variants thereof.

Preferably, a suitable variant for use as described herein comprises anantibody antigen binding site comprising a VH domain and a VL domain ofany one of antibodies F8, H1, B2, C5, D5, E5, C8, F1, B7, E8 and G9,wherein the valine (V) residue at position 5 of the VH domain issubstituted with leucine (L), and/or the lysine (K) residue at position18 of the VL domain is substituted with arginine (R). Most preferably, asuitable variant for use as described herein comprises an antibodyantigen binding site comprising the F8 VH V5L domain of SEQ ID NO: 16and the F8 VL K18R domain of SEQ ID NO: 78, wherein the valine (V)residue at position 5 of the VH domain is substituted by leucine (L),and/or the lysine (K) residue at position 18 of the VL domain issubstituted by arginine (R).

A specific binding member for use in the invention may comprise a set ofH and/or L CDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 orG9, or a set of H and/or L CDRs of antibody Hi, B2, C5, D5, E5, C8, F8,F1, B7, E8 or G9 with ten or fewer, e.g. one, two, three, four, or five,amino acid substitutions within the disclosed set of H and/or L CDRs.Preferably, a specific binding member for use in the invention comprisesa set of H and/or L CDRs of antibody B2, C5, D5, C8, F8, B7 or G9 withten or fewer, e.g. one, two, three, four, or five, amino acidsubstitutions within the disclosed set of H and/or L CDRs. Preferably, aspecific binding member for use in the invention comprises a set of Hand/or L CDRs of antibody F8 with ten or fewer, e.g. one, two, three,four, or five, amino acid substitutions within the disclosed set of Hand/or L CDRs.

Substitutions may potentially be made at any residue within the set ofCDRs, and may be within CDR1, CDR2 and/or CDR3.

For example, a specific binding member for use in the invention maycomprise one or more CDRs as described herein, e.g. a CDR3, andoptionally also a CDR1 and CDR2 to form a set of CDRs.

A specific binding member for use in the invention may also comprise anantibody molecule, e.g. a human antibody molecule. The specific bindingmember normally comprises an antibody VH and/or VL domain. VH domains ofspecific binding members are also provided for use in the invention.Within each of the VH and VL domains are complementarity determiningregions, (“CDRs”), and framework regions, (“FRs”). A VH domain comprisesa set of HCDRs, and a VL domain comprises a set of LCDRs. An antibodymolecule may comprise an antibody VH domain comprising a VH CDR1, CDR2and CDR3 and a framework. It may alternatively or also comprise anantibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework.The VH and VL domains and CDRs of antibodies H1, B2, C5, D5, E5, C8, F8(and its variant comprising V5L VH and K18R VL), F1, B7, E8 and G9 aredescribed herein. All VH and VL sequences, CDR sequences, sets of CDRsand sets of HCDRs and sets of LCDRs disclosed herein representembodiments of a specific binding member for use in the invention. Asdescribed herein, a “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, aset of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refersto LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs”includes HCDRs and LCDRs.

A specific binding member for use in the invention may comprise anantibody VH domain comprising complementarity determining regions HCDR1,HCDR2 and HCDR3 and a framework, wherein HCDR1 is SEQ ID NO: 30, 32, 34,36, 38, 40, 42, 44, 46, 48, or 50, and wherein optionally HCDR2 is SEQID NO: 56, and/or HCDR3 is SEQ ID NO: 60. Preferably, the HCDR1 is SEQID NO: 42.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below, a VH or VLdomain alone may be used to bind antigen. Thus, a specific bindingmember for use in the invention may further comprise an antibody VLdomain comprising complementarity determining regions LCDR1, LCDR2 andLCDR3 and a framework, wherein LCDR1 is SEQ ID NO: 90, 92, 94, 96, 98,100, 102, 104, 106, 108, or 110, and wherein optionally LCDR2 is SEQ IDNO: 114 and/or LCDR3 is SEQ ID NO: 118. Preferably, the LCDR1 is SEQ IDNO: 102.

A specific binding member for use in the invention may be an isolatedantibody molecule for the ED-A of fibronectin, comprising a VH domainand a VL domain, wherein the VH domain comprises a framework and a setof complementarity determining regions HCDR1, HCDR2 and HCDR3 andwherein the VL domain comprises complementarity determining regionsLCDR1, LCDR2 and LCDR3 and a framework, and wherein

-   HCDR1 has amino acid sequence SEQ ID NO: 30, 32, 34, 36, 38, 40, 42,    44, 46, 48, or 50;-   HCDR2 has amino acid sequence SEQ ID NO: 56;-   HCDR3 has amino acid sequence SEQ ID NO: 60;-   LCDR1 has amino acid sequence SEQ ID NO: 90, 92, 94, 96, 98, 100,    102, 104, 106, 108, or 110;-   LCDR2 has amino acid sequence SEQ ID NO: 114; and-   LCDR3 has amino acid sequence SEQ ID NO: 118.

Preferably, the HCDR1 is SEQ ID NO: 42, and the LCDR1 is SEQ ID NO: 102.

One or more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule for usein the invention. Framework regions may comprise human germline genesegment sequences. Thus, the framework may be germlined, whereby one ormore residues within the framework are changed to match the residues atthe equivalent position in the most similar human germline framework. Aspecific binding member for use in the invention may be an isolatedantibody molecule having a VH domain comprising a set of HCDRs in ahuman germline framework, e.g. DP47. Normally the specific bindingmember also has a VL domain comprising a set of LCDRs, e.g. in a humangermline framework. The human germline framework of the VL domain may beDPK22.

A VH domain for use in the invention may have amino acid sequence SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24.

Preferably, a VH domain for use in the invention has amino acid sequenceSEQ ID NO: 14 or 16. A VL domain for use in the invention may have theamino acid SEQ ID NO: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, or 86.Preferably, a VL domain for use in the invention has amino acid SEQ IDNO: 76 or 78.

A specific binding member for use in the invention may be or comprise asingle chain Fv (scFv), comprising a VH domain and a VL domain joinedvia a peptide linker. The skilled person may select an appropriatelength and sequence of linker, e.g. at least 5 or at least 10 aminoacids in length, up to about 15, up to about 20 or up to about 25 aminoacids in length. The linker may have the amino acid sequence GSSGG (SEQID NO: 122).

The specific binding member may be a diabody, which is a multivalent ormultispecific fragment constructed by gene fusion (WO94/13804; Holliger1993a).

A single chain Fv (scFv) may be comprised within a mini-immunoglobulinor small immunoprotein (SIP), e.g. as described in (Li et al., 1997). AnSIP may comprise an scFv molecule fused to the CH4 domain of the humanIgE secretory isoform IgE-S2 (ε_(s2)-CH4; Batista et al., 1996) formingan homo-dimeric mini-immunoglobulin antibody molecule.

Alternatively, a specific binding member for use in the invention maycomprise an antigen-binding site within a non-antibody molecule,normally provided by one or more CDRs e.g. a set of CDRs in anon-antibody protein scaffold. Specific binding members, includingnon-antibody and antibody molecules, are described in more detailelsewhere herein.

According to a fifth aspect, the invention provides a specific bindingmember that binds tenascin-C for use in a method of treatment ofendometriosis, psoriatic arthritis or psoriasis. The invention alsoprovides use of a specific binding member that binds tenascin-C for themanufacture of a medicament for treating endometriosis, psoriaticarthritis or psoriasis. The invention also provides a method of treatingendometriosis, psoriatic arthritis or psoriasis in a patient, the methodcomprising administering to a patient a therapeutically effective amountof a medicament comprising a specific binding member which bindstenascin-C. Preferably, the specific binding member binds humantenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thisfifth aspect of the invention may bind specifically to tenascin-C largeisoform. For example, the specific binding member may bindpreferentially to tenascin-C large isoform relative to tenascin-C smallisoform. Preferably, the specific binding member binds to the A1, A2,A3, A4, B and/or D domain of tenascin-C large isoform. Most preferably,the specific binding member binds to the A1 domain of tenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thisfifth aspect of the invention, may be conjugated to a detectable label,a radioisotope, or a bioactive molecule, such as a cytokine, hormone, atherapeutic isotope, or a cytotoxic drug. For example, the specificbinding member may be conjugated to a cytokine, such as IL-10, TGF-β,IL-2, IL-12, IL-15, IL-21, IL-24, IL-33, tumour necrosis factor (TNF),or interferon-α, -β or -γ. The specific binding member may be conjugatedto the bioactive molecule by a cleavable linker.

According to a sixth aspect, the invention provides a specific bindingmember that binds tenascin-C, for use in the delivery to theneovasculature of endometriotic, psoriatic arthritic or psoriatic tissueof a molecule conjugated to the specific binding member. The inventionalso provides use of a specific binding member that binds tenascin-C forthe manufacture of a medicament for delivery of a molecule conjugated tothe specific binding member to the neovasculature of endometriotic,psoriatic arthritic or psoriatic tissue. The invention also provides amethod of delivering a molecule to the neovasculature of endometriotic,psoriatic arthritic or psoriatic tissue in a human or animal, whereinthe molecule is conjugated to a specific binding member which bindstenascin-C to form a conjugate and the method comprises administeringthe conjugate to the human or animal. Preferably, the specific bindingmember binds human tenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thissixth aspect of the invention may bind specifically to tenascin-C largeisoform. For example, the specific binding member may bindpreferentially to tenascin-C large isoform relative to tenascin-C smallisoform. Preferably, the specific binding member binds to the A1, A2,A3, A4, B and/or D domain of tenascin-C large isoform. Most preferably,the specific binding member binds to the A1 domain of tenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thissixth aspect of the invention, may be conjugated to a detectable label,a radioisotope, or a bioactive molecule, such as a cytokine, hormone, atherapeutic isotope, or a cytotoxic drug. For example, the specificbinding member may be conjugated to a cytokine, such as IL-10, TGF-β,IL-2, IL-12, IL-15, IL-21, IL-24, IL-33, tumour necrosis factor (TNF),or interferon-α, -β or -γ. The specific binding member may be conjugatedto the bioactive molecule by a cleavable linker.

According to a seventh aspect, the invention provides a specific bindingmember that binds tenascin-C for use in a method of diagnosis ofendometriosis, psoriatic arthritis or psoriasis. The invention alsoprovides use of a specific binding member that binds tenascin-C for themanufacture of a diagnostic product for diagnosing endometriosis,psoriatic arthritis or psoriasis. The invention also provides a methodof detecting or diagnosing endometriosis, psoriatic arthritis orpsoriasis in a human or animal, wherein the method comprises the stepsof:

-   -   (a) administering to the human or animal a specific binding        member which binds tenascin-C, and    -   (b) determining the presence or absence of the specific binding        member in sites of endometriosis, psoriatic arthritis or        psoriasis of the human or animal body;    -   wherein localisation of the specific binding member to sites of        endometriosis, psoriatic arthritis or psoriasis indicates the        presence of endometriosis, psoriatic arthritis or psoriasis.

Preferably, the specific binding member binds human tenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thisseventh aspect of the invention may bind specifically to tenascin-Clarge isoform. For example, the specific binding member may bindpreferentially to tenascin-C large isoform relative to tenascin-C smallisoform. Preferably, the specific binding member binds to the A1, A2,A3, A4, B and/or D domain of tenascin-C large isoform. Most preferably,the specific binding member binds to the A1 domain of tenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thisseventh aspect of the invention, may be conjugated to a detectablelabel, or a radioisotope.

In an eighth aspect, the invention provides a specific binding memberthat binds tenascin-C for use in a method of imaging endometriotic,psoriatic arthritic or psoriatic tissue. The invention also provides useof a specific binding member that binds tenascin-C for the manufactureof an imaging agent for imaging endometriotic, psoriatic arthritic orpsoriatic tissue. The invention also provides a method of detecting orimaging endometriotic, psoriatic arthritic or psoriatic tissue in ahuman or animal, wherein the method comprises the steps of:

-   -   (a) administering to the human or animal a specific binding        member which binds tenascin-C, and    -   (b) detecting the binding of the specific binding member to        endometriotic, psoriatic arthritic or psoriatic tissue in the        human or animal body.

Preferably, the specific binding member binds human tenascin-C. Thespecific binding member, e.g. an antibody molecule, for use in thiseighth aspect of the invention may bind specifically to tenascin-C largeisoform. For example, the specific binding member may bindpreferentially to tenascin-C large isoform relative to tenascin-C smallisoform. Preferably, the specific binding member binds to the A1, A2,A3, A4, B and/or D domain of tenascin-C large isoform. Most preferably,the specific binding member binds to the A1 domain of tenascin-C.

The specific binding member, e.g. an antibody molecule, for use in thiseighth aspect of the invention, may be conjugated to a detectable label,or a radioisotope.

A specific binding member for use in the invention may be an antibodywhich binds tenascin-C, and/or the A1, A2, A3, A4, B and/or D domain ofthe tenascin-C large isoform, wherein the antibody comprises one or morecomplementarity determining regions (CDRs) of antibody F16 or 4A1-F16(Brack et al., 12, 3200-3208, (2006) Clin. Cancer Res.), or variantsthereof.

A specific binding member for use in the invention may comprise a set ofH and/or L CDRs of antibody F16 or 4A1-F16, or a set of H and/or L CDRsof antibody F16 or 4A1-F16 with ten or fewer, e.g. one, two, three,four, or five, amino acid substitutions within the disclosed set of Hand/or L CDRs. Preferably, a specific binding member for use in theinvention comprises a set of H and/or L CDRs of antibody F16 or 4A1-F16with ten or fewer, e.g. one, two, three, four, or five, amino acidsubstitutions within the disclosed set of H and/or L CDRs. Preferably, aspecific binding member for use in the invention comprises a set of Hand/or L CDRs of antibody F16 or 4A1-F16 with ten or fewer, e.g. one,two, three, four, or five, amino acid substitutions within the disclosedset of H and/or L CDRs.

Substitutions may potentially be made at any residue within the set ofCDRs, and may be within CDR1, CDR2 and/or CDR3.

For example, a specific binding member for use in the invention maycomprise one or more CDRs as described herein, e.g. a CDR3, andoptionally also a CDR1 and CDR2 to form a set of CDRs.

A specific binding member for use in the invention may also comprise anantibody molecule, e.g. a human antibody molecule. The specific bindingmember normally comprises an antibody VH and/or VL domain. VH domains ofspecific binding members are also provided for use in the invention.Within each of the VH and VL domains are complementarity determiningregions, (“CDRs”), and framework regions, (“FRs”). A VH domain comprisesa set of HCDRs, and a VL domain comprises a set of LCDRs. An antibodymolecule may comprise an antibody VH domain comprising a VH CDR1, CDR2and CDR3 and a framework. It may alternatively or also comprise anantibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework.The VH and VL domains and CDRs of antibody F16 and 4A1-F16 are describedherein. All VH and VL sequences, CDR sequences, sets of CDRs and sets ofHCDRs and sets of LCDRs disclosed herein represent embodiments of aspecific binding member for use in the invention. As described herein, a“set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refersto HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 andLCDR3. Unless otherwise stated, a “set of CDRs” includes HCDRs andLCDRs.

A specific binding member for use in the invention may comprise anantibody VH domain comprising complementarity determining regions HCDR1,HCDR2 and HCDR3 and a framework, wherein HCDR1 is SEQ ID NO: 52 or 54,and wherein optionally HCDR2 is SEQ ID NO: 58, and/or HCDR3 is SEQ IDNO: 62. Preferably, the HCDR1 is SEQ ID NO: 52.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below, a VH or VLdomain alone may be used to bind antigen. Thus, a specific bindingmember for use in the invention may further comprise an antibody VLdomain comprising complementarity determining regions LCDR1, LCDR2 andLCDR3 and a framework, wherein LCDR1 is SEQ ID NO: 112, and whereinoptionally LCDR2 is SEQ ID NO: 116, and/or LCDR3 is SEQ ID NO: 120.

A specific binding member for use in the invention may be an isolatedantibody molecule for the A1 domain of tenascin-C, comprising a VHdomain and a VL domain, wherein the VH domain comprises a framework anda set of complementarity determining regions HCDR1, HCDR2 and HCDR3 andwherein the VL domain comprises complementarity determining regionsLCDR1, LCDR2 and LCDR3 and a framework, and wherein

-   HCDR1 has amino acid sequence SEQ ID NO: 52 or 54;-   HCDR2 has amino acid sequence SEQ ID NO: 58;-   HCDR3 has amino acid sequence SEQ ID NO: 62;-   LCDR1 has amino acid sequence SEQ ID NO: 112;-   LCDR2 has amino acid sequence SEQ ID NO: 116; and-   LCDR3 has amino acid sequence SEQ ID NO: 120.

One or more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule for usein the invention. Framework regions may comprise human germline genesegment sequences. Thus, the framework may be germlined, whereby one ormore residues within the framework are changed to match the residues atthe equivalent position in the most similar human germline framework. Aspecific binding member for use in the invention may be an isolatedantibody molecule having a VH domain comprising a set of HCDRs in ahuman germline framework, e.g. DP47. Normally the specific bindingmember also has a VL domain comprising a set of LCDRs, e.g. in a humangermline framework. The human germline framework of the VL domain may beDPK22.

A VH domain for use in the invention may have amino acid sequence SEQ IDNO: 26 or 28. Preferably, the VH domain has the amino acid sequence SEQID NO: 26. A VL domain for use in the invention 5 may have the aminoacid SEQ ID NO: 88.

The VL domain of F16 and 4A1-F16 (SEQ ID NO: 88) may optionally includean extra glycine residue at its C-terminal end.

A specific binding member for use in the invention may be or comprise asingle chain Fv (scFv), comprising a VH domain and a VL domain joinedvia a peptide linker. The skilled person may select an appropriatelength and sequence of linker, e.g. at least 5 or at least 10 aminoacids in length, up to about 15, up to about 20 or up to about 25 aminoacids in length. The linker may have the amino acid sequence GSSGG (SEQID NO: 122).

The specific binding member may be a diabody, which is a multivalent ormultispecific fragment constructed by gene fusion (WO94/13804; Holliger1993a).

A single chain Fv (scFv) may be comprised within a mini-immunoglobulinor small immunoprotein (SIP), e.g. as described in (Li et al., 1997). AnSIP may comprise an scFv molecule fused to the CH4 domain of the humanIgE secretory isoform IgE-S2 (ε_(s2)-CH4; Batista et al., 1996) formingan homo-dimeric mini-immunoglobulin antibody molecule.

Alternatively, a specific binding member for use in the invention maycomprise an antigen-binding site within a non-antibody molecule,normally provided by one or more CDRs e.g. a set of CDRs in anon-antibody protein scaffold. Specific binding members, includingnon-antibody and antibody molecules, are described in more detailelsewhere herein.

These and other aspects of the invention are described in further detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of immunohistochemistry on samples frompatients with endometriosis using antibodies directed to markers ofangiogenesis. Darker staining indicates strong expression of theantigen, visualized by black arrows. Areas of negative staining aroundperivascular structures are indicated using white arrows. A, B and Cshow staining with F8-SIP, which is an antibody molecule that bindsED-A, disclosed herein. D, E and F show staining with L19-SIP, which isan antibody molecule that binds ED-B (e.g. Pini et al. 1998). G, H and Ishow staining with F16-SIP, which is an antibody molecule that binds theA1 domain of Tenascin-C (WO2006/050834). J, K and L are negativecontrols in which no primary antibody was added. A, D, G, J, B, E, H andK show samples viewed under 10× magnification and C, F, I and L showsamples viewed under 5× magnification.

FIG. 2 shows the results of immunohistochemistry on samples frompatients with ulcerative colitis using antibodies directed to markers ofangiogenesis. The white arrows indicate areas of negative stainingaround perivascular structures. FIG. 2A shows staining with F8-SIP,which is an antibody molecule that binds ED-A, disclosed herein. FIG. 2Bshows staining with L19-SIP, which is an antibody molecule that bindsED-B (e.g. Pini et al. 1998). FIG. 2C shows staining with F16-SIP, whichis an antibody molecule that binds the A1 domain of Tenascin-C(WO2006/050834). FIG. 2D is a negative control in which no primaryantibody was added.

FIG. 3 shows the results of immunohistochemistry on samples frompatients with psoriatic arthritis using antibodies directed to markersof angiogenesis. Darker staining indicates strong expression of theantigen, visualized by black arrows. Areas of negative staining aroundperivascular structures are indicated using white arrows. FIG. 3A showsstaining with F8-SIP, which is an antibody molecule that binds ED-A,disclosed herein. FIG. 3B shows staining with L19-SIP, which is anantibody molecule that binds ED-B (e.g. Pini et al. 1998). FIG. 3C showsstaining with F16-SIP, which is an antibody molecule that binds the A1domain of Tenascin-C (WO2006/050834). FIG. 3D is a negative control inwhich no primary antibody was added.

FIG. 4 shows the results of immunohistochemistry on samples frompatients with multiple sclerosis using antibodies directed to markers ofangiogenesis. The white arrows indicate areas of negative stainingaround perivascular structures. FIG. 4A shows staining with F8-SIP,which is an antibody molecule that binds ED-A, disclosed herein. FIG. 4Bshows staining with L19-SIP, which is an antibody molecule that bindsED-B (e.g. Pini et al. 1998). FIG. 4C shows staining with F16-SIP, whichis an antibody molecule that binds the A1 domain of Tenascin-C(WO2006/050834). FIG. 4D is a negative control in which no primaryantibody was added.

FIG. 5 shows the results of immunohistochemistry on samples frompatients with psoriasis using antibodies directed to markers ofangiogenesis. Darker staining indicates strong expression of theantigen, visualized by black arrows. Areas of negative staining aroundperivascular structures are indicated using white arrows. FIG. 5A showsstaining with F8-SIP, which is an antibody molecule that binds ED-A,disclosed herein.

FIG. 58 shows staining with L19-SIP, which is an antibody molecule thatbinds ED-B (e.g. Pini et al. 1998). FIG. 5C shows staining with F16-SIP,which is an antibody molecule that binds the A1 domain of Tenascin-C(WO2006/050834). FIG. 5D is a negative control in which no primaryantibody was added.

FIG. 6A shows the nucleotide sequences of the anti-ED-A antibody F8heavy chain (VH) (SEQ ID NO: 13) and its variant, F8 V5L VH (SEQ ID NO:15). The nucleotide sequence of the heavy chain CDR1 (SEQ ID NO: 41) ofanti-ED-A antibody F8 is underlined. The nucleotide sequence of theheavy chain CDR2 (SEQ ID NO: 55) of the anti-ED-A antibody F8 is shownin italics and underlined. The nucleotide sequence of the heavy chainCDR3 (SEQ ID NO: 59) of anti-ED-A antibody F8 is shown in bold andunderlined.

FIG. 6B shows the nucleotide sequence of the anti-ED-A antibody F8linker sequence (SEQ ID NO: 121).

FIG. 6C shows the nucleotide sequences of the anti-ED-A antibody F8light chain (VL) (SEQ ID NO: 75) and its variant, F8 K18R VL (SEQ ID NO:77). The nucleotide sequence of the light chain CDR1 (SEQ ID NO: 101) ofanti-ED-A antibody F8 is underlined. The nucleotide sequence of thelight chain CDR2 (SEQ ID NO: 113) of the anti-ED-A antibody F8 is shownin italics and underlined. The nucleotide sequence of the light chainCDR3 (SEQ ID NO: 117) of anti-ED-A antibody F8 is shown in bold andunderlined.

FIG. 7A shows the amino acid sequences of the anti-ED-A antibody F8heavy chain (VH) (SEQ ID NO: 14), and its variant, F8 V5L VH (SEQ ID NO:16). The amino acid sequence of the heavy chain CDR1 (SEQ ID NO: 42) ofanti-ED-A antibody F8 is underlined. The amino acid sequence of theheavy chain CDR2 (SEQ ID NO: 56) of the anti-ED-A antibody F8 is shownin italics and underlined. The amino acid sequence of the heavy chainCDR3 (SEQ ID NO: 60) of anti-ED-A antibody F8 is shown in bold andunderlined.

FIG. 7B shows the amino acid sequence of the anti-ED-A antibody F8linker sequence (SEQ ID NO: 122).

FIG. 7C shows the amino acid sequences of the anti-ED-A antibody F8light chain (VL) (SEQ ID NO: 76) and its variant, F8 K18R VL (SEQ ID NO:78). The amino acid sequence of the light chain CDR1 (SEQ ID NO: 102) ofanti-ED-A antibody F8 is underlined. The amino acid sequence of thelight chain CDR2 (SEQ ID NO: 114) of the anti-ED-A antibody F8 is shownin italics and underlined. The amino acid sequence of the light chainCDR3 (SEQ ID NO: 118) of anti-ED-A antibody F8 is shown in bold andunderlined.

FIG. 8A shows the nucleotide sequences of the heavy chain (VH) of theanti-tenascin-C antibody F16 (SEQ ID NO: 25) and its variant, 4A1-F16(SEQ ID NO: 27). The nucleotide sequences of the heavy chain CDR1 ofanti-tenascin-C antibodies F16 and F16-4A1 are underlined (SEQ ID NOs:51 and 53 respectively). The nucleotide sequence of the heavy chain CDR2(SEQ ID NO: 57) of the anti-tenascin-C antibodies F16 and F16-4A1 isshown in italics and underlined. The nucleotide sequence of the heavychain CDR3 (SEQ ID NO: 61) of the anti-tenascin-C antibodies F16 andF16-4A1 is shown in bold and underlined.

FIG. 8B shows the nucleotide sequence of the linker sequence (SEQ ID NO:121) of the anti-tenascin-C antibodies F16 and F16-4A1.

FIG. 8C shows the nucleotide sequence of the light chain (VL) (SEQ IDNO: 87) of the anti-tenascin-C antibodies F16 and F16-4A1. Thenucleotide sequence of the light chain CDR1 (SEQ ID NO: 111) of theanti-tenascin-C antibodies F16 and F16-4A1 is underlined. The nucleotidesequence of the light chain CDR2 (SEQ ID NO: 115) of the anti-tenascin-Cantibodies F16 and F16-4A1 is shown in italics and underlined. Thenucleotide sequence of the light chain CDR3 (SEQ ID NO: 119) ofanti-tenascin-C antibodies F16 and F16-4A1 is shown in bold andunderlined.

FIG. 9A shows the amino acid sequence of the heavy chain (VH) of theanti-tenascin-C antibody F16 (SEQ ID NO: 26) and its variant, 4A1-F16(SEQ ID NO: 28). The amino acid sequence of the heavy chain CDR1 of theanti-tenascin-C antibody F16 (SEQ ID NO: 52) and 4A1-F16 (SEQ ID NO: 54)is underlined. The amino acid sequence of the heavy chain CDR2 (SEQ IDNO: 58) of the anti-tenascin-C antibodies F16 and 4A1-F16 is shown initalics and underlined. The amino acid sequence of the heavy chain CDR3(SEQ ID NO: 62) of anti-tenascin-C antibodies F16 and 4A1-F16 is shownin bold and underlined.

FIG. 9B shows the amino acid sequence of the anti-tenascin-C antibodyF16 linker sequence (SEQ ID NO: 122).

FIG. 9C shows the amino acid sequence of the light chain (VL) (SEQ IDNO: 88) of the anti-tenascin-C antibodies F16 and 4A1-F16. The aminoacid sequence of the light chain CDR1 (SEQ ID NO: 112) ofanti-tenascin-C antibodies F16 and 4A1-F16 is underlined. The amino acidsequence of the light chain CDR2 (SEQ ID NO: 116) of the anti-tenascin-Cantibodies F16 and 4A1-F16 is shown in italics and underlined. The aminoacid sequence of the light chain CDR3 (SEQ ID NO: 120) ofanti-tenascin-C antibodies F16 and 4A1-F16 is shown in bold andunderlined.

FIG. 10 shows the results of near infrared imaging of endometriosislesions in mice. Areas of endometriosis in the mice are indicated bycircles. FIGS. 10 A and B show the results of near infrared imagingusing a SIP(F8)-ALEXA750 antibody molecule, which binds ED-A.SIP(F8)-ALEXA750 was injected into mice with endometriosis and imaged 24h after injection. Arrows indicate areas of positive imaging. FIG. 10Cshows the results of near-infrared imaging using a SIP(F16)-ALEXA750antibody molecule which recognises the human A1 domain of tenascin-C.SIP(F16)-ALEXA750 was injected into mice with endometriosis and imaged24 h after injection.

FIG. 11 shows the results of ex vivo staining of murine endometriosislesions. FIGS. 11 A, B and C show immunofluorescent detection ofSIP(F8)-ALEXA750 using a rabbit anti-human IgE antibody followed by goatanti-rabbit IgG. FIGS. 11 D, E and F show detection of blood vesselsusing a rat anti-CD31 antibody followed by donkey anti-rat IgG. FIGS. 11G, H and I represent negative controls and show the results of probingthe lesions with goat anti-rabbit IgG alone.

TERMINOLOGY

Fibronectin

Fibronectin is an antigen subject to alternative splicing, and a numberof alternative isoforms of fibronectin are known, as described elsewhereherein. Extra Domain-A (EDA or ED-A) is also known as ED, extra type IIIrepeat A (EIIIA) or EDI. The sequence of human ED-A has been publishedby Kornblihtt et al. (1984), Nucleic Acids Res. 12, 5853-5868 andPaolella et al. (1988), Nucleic Acids Res. 16, 3545-3557. The sequenceof human ED-A is also available on the SwissProt database as amino acids1631-1720 (Fibronectin type-III 12; extra domain 2) of the amino acidsequence deposited under accession number P02751. The sequence of mouseED-A is available on the SwissProt database as amino acids 1721-1810(Fibronectin type-III 13; extra domain 2) of the amino acid sequencedeposited under accession number P11276.

The ED-A isoform of fibronectin (A-FN) contains the Extra Domain-A(ED-A). The sequence of the human A-FN can be deduced from thecorresponding human fibronectin precursor sequence which is available onthe SwissProt database under accession number P02751. The sequence ofthe mouse A-FN can be deduced from the corresponding mouse fibronectinprecursor sequence which is available on the SwissProt database underaccession number P11276. The A-FN may be the human ED-A isoform offibronectin. The ED-A may be the Extra Domain-A of human fibronectin.

ED-A is a 90 amino acid sequence which is inserted into fibronectin (FN)by alternative splicing and is located between domain 11 and 12 of FN(Borsi et al., 1987, J. Cell Biol., 104, 595-600). ED-A is mainly absentin the plasma form of FN but is abundant during embryogenesis, tissueremodelling, fibrosis, cardiac transplantation and solid tumour growth.

Alternative Splicing

Alternative splicing refers to the occurrence of different patterns ofsplicing of a primary RNA transcript of DNA to produce different mRNAs.After excision of introns, selection may determine which exons arespliced together to form the mRNA. Alternative splicing leads toproduction of different isoforms containing different exons and/ordifferent numbers of exons. For example one isoform may comprise anadditional amino acid sequence corresponding to one or more exons, whichmay comprise one or more domains.

Tenascin-C

Tenascin-C is a large hexameric glycoprotein of the extracellular matrixwhich modulates cellular adhesion. It is involved in processes such ascell proliferation and cell migration and is associated with changes intissue architecture as occurring during morphogenesis and embryogenesisas well as under tumorigenesis or angiogenesis.

A strong over-expression of the large isoform of tenascin-C has beenreported for a number of tumors [Borsi 1992 supra], and monoclonalantibodies specific for domains A1 and D, respectively, have beenextensively characterised in the clinic [Riva et al., Int J Cancer(1992) 51:7-13; Riva et al., Cancer Res (1995), 55, 5952s-5956s;Paganelli et al., Eur J Nucl Med (1994) 21, 314-321. Reardon et al., JClin Oncol (2002), 20, 1389-1397; Signer et al., J Clin Oncol (1998) 16,2202-2212].

Human monoclonal antibody fragments specific to tenascin-C are describedin WO2006/050834 and shown to bind preferentially to tumor tissuerelative to normal tissue. These antibodies are useful, for example, indelivering toxins, such as cytokines, specifically to tumour cells.

Specific Binding Member

This describes one member of a pair of molecules that bind specificallyto one another. The members of a specific binding pair may be naturallyderived or wholly or partially synthetically produced. One member of thepair of molecules has an area on its surface, or a cavity, which bindsto and is therefore complementary to a particular spatial and polarorganization of the other member of the pair of molecules. Examples oftypes of binding pairs are antigen-antibody, biotin-avidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate. The presentinvention is concerned with antigen-antibody type reactions.

A specific binding member normally comprises a molecule having anantigen-binding site. For example, a specific binding member may be anantibody molecule or a non-antibody protein that comprises anantigen-binding site.

An antigen binding site may be provided by means of arrangement ofcomplementarity determining regions (CDRs) on non-antibody proteinscaffolds such as fibronectin or cytochrome B etc. (Haan & Maggos, 2004;Koide 1998; Nygren 1997), or by randomising or mutating amino acidresidues of a loop within a protein scaffold to confer bindingspecificity for a desired target. Scaffolds for engineering novelbinding sites in proteins have been reviewed in detail by Nygren et al.(1997). Protein scaffolds for antibody mimics are disclosed inWO/0034784, which is herein incorporated by reference in its entirety,in which the inventors describe proteins (antibody mimics) that includea fibronectin type III domain having at least one randomised loop. Asuitable scaffold into which to graft one or more CDRs, e.g. a set ofHCDRs, may be provided by any domain member of the immunoglobulin genesuperfamily. The scaffold may be a human or non-human protein. Anadvantage of a non-antibody protein scaffold is that it may provide anantigen-binding site in a scaffold molecule that is smaller and/oreasier to manufacture than at least some antibody molecules. Small sizeof a binding member may confer useful physiological properties such asan ability to enter cells, penetrate deep into tissues or reach targetswithin other structures, or to bind within protein cavities of thetarget antigen. Use of antigen binding sites in non-antibody proteinscaffolds is reviewed in Wess, 2004. Typical are proteins having astable backbone and one or more variable loops, in which the amino acidsequence of the loop or loops is specifically or randomly mutated tocreate an antigen-binding site that binds the target antigen. Suchproteins include the IgG-binding domains of protein A from S. aureus,transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type IIIdomain) and lipocalins. Other approaches include synthetic “Microbodies”(Selecore GmbH), which are based on cyclotides—small proteins havingintra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, aspecific binding member for use in the present invention may compriseother amino acids, e.g. forming a peptide or polypeptide, such as afolded domain, or to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. Binding membersfor use in the invention may carry a detectable label, or may beconjugated to a toxin or a targeting moiety or enzyme (e.g. via apeptidyl bond or linker). For example, a binding member may comprise acatalytic site (e.g. in an enzyme domain) as well as an antigen bindingsite, wherein the antigen binding site binds to the antigen and thustargets the catalytic site to the antigen. The catalytic site mayinhibit biological function of the antigen, e.g. by cleavage.

Although, as noted, CDRs can be carried by non-antibody scaffolds, thestructure for carrying a CDR or a set of CDRs will generally be anantibody heavy or light chain sequence or substantial portion thereof inwhich the CDR or set of CDRs is located at a location corresponding tothe CDR or set of CDRs of naturally occurring VH and VL antibodyvariable domains encoded by rearranged immunoglobulin genes. Thestructures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat 1987, and updates thereof, nowavailable on the Internet (at immuno.bme.nwu.edu or find “Kabat” usingany search engine).

By CDR region or CDR, it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al. (1987), (Kabat 1991a, and later editions). An antibodytypically contains 3 heavy chain CDRs and 3 light chain CDRs. The termCDR or CDRs is used here in order to indicate, according to the case,one of these regions or several, or even the whole, of these regionswhich contain the majority of the amino acid residues responsible forthe binding by affinity of the antibody for the antigen or the epitopewhich it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It can be as short as 2 amino acids although the longest size knownis 26. Functionally, HCDR3 plays a role in part in the determination ofthe specificity of the antibody (Segal 1974; Amit 1986; Chothia 1987;Chothia 1989; Caton 1990; Sharon 1990a; Sharon 1990b; Kabat et al.,1991b).

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also relates to any polypeptide orprotein comprising an antibody antigen-binding site. It must beunderstood here that the invention does not relate to the antibodies innatural form, that is to say they are not in their natural environmentbut that they have been able to be isolated or obtained by purificationfrom natural sources, or else obtained by genetic recombination, or bychemical synthesis, and that they can then contain unnatural amino acidsas will be described later. Antibody fragments that comprise an antibodyantigen-binding site include, but are not limited to, antibody moleculessuch as Fab, Fab′, Fab′-SH, scFv, Fv, dAb, Fd; and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that bind the target antigen. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe CDRs, of an antibody to the constant regions, or constant regionsplus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body ofsubsequent literature. A hybridoma or other cell producing an antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any binding member orsubstance having an antibody antigen-binding site with the requiredspecificity and/or binding to antigen. Thus, this term covers antibodyfragments and derivatives, including any polypeptide comprising anantibody antigen-binding site, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an antibody antigen-bindingsite, or equivalent, fused to another polypeptide (e.g. derived fromanother species or belonging to another antibody class or subclass) aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023, and a large body ofsubsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann & Dubel (2001).Phage display, another established technique for generating bindingmembers has been described in detail in many publications such asWO92/01047 (discussed further below) and U.S. Pat. No. 5,969,108, U.S.Pat. No. 5,565,332, U.S. Pat. No. 5,733,743, U.S. Pat. No. 5,858,657,U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,872,215, U.S. Pat. No.5,885,793, U.S. Pat. No. 5,962,255, U.S. Pat. No. 6,140,471, U.S. Pat.No. 6,172,197, U.S. Pat. No. 6,225,447, U.S. Pat. No. 6,291,650, U.S.Pat. No. 6,492,160, U.S. Pat. No. 6,521,404 and Kontermann & Dubel(2001). Transgenic mice in which the mouse antibody genes areinactivated and functionally replaced with human antibody genes whileleaving intact other components of the mouse immune system, can be usedfor isolating human antibodies (Mendez 1997).

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.(2000) or Krebs et al. (2001).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward 1989; McCafferty 1990; Holt 2003), which consists of a VHor a VL domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird 1988; Huston 1988); (viii) bispecific singlechain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; Holliger1993a). Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter 1996). Minibodies comprising a scFv joined to a CH3 domain mayalso be made (Hu 1996). Other examples of binding fragments are Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

Antibody fragments for use in the invention can be obtained startingfrom any of the antibody molecules described herein, e.g. antibodymolecules comprising VH and/or VL domains or CDRs of any of antibodiesdescribed herein, by methods such as digestion by enzymes, such aspepsin or papain and/or by cleavage of the disulfide bridges by chemicalreduction. In another manner, antibody fragments of the presentinvention may be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc., or by nucleicacid synthesis and expression.

Functional antibody fragments according to the present invention includeany functional fragment whose half-life is increased by a chemicalmodification, especially by PEGylation, or by incorporation in aliposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain (Holt 2003). VH dAbs occur naturally in camelids (e.g. camel,llama) and may be produced by immunizing a camelid with a targetantigen, isolating antigen-specific B cells and directly cloning dAbgenes from individual B cells. dAbs are also producible in cell culture.Their small size, good solubility and temperature stability makes themparticularly physiologically useful and suitable for selection andaffinity maturation. A binding member of the present invention may be adAb comprising a VH or VL domain substantially as set out herein, or aVH or VL domain comprising a set of CDRs substantially as set outherein.

As used herein, the phrase “substantially as set out” refers to thecharacteristic(s) of the relevant CDRs of the VH or VL domain of bindingmembers described herein will be either identical or highly similar tothe specified regions of which the sequence is set out herein. Asdescribed herein, the phrase “highly similar” with respect to specifiedregion(s) of one or more variable domains, it is contemplated that from1 to about 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4,amino acid substitutions may be made in the CDR and/or VH or VL domain.

Bispecific or bifunctional antibodies form a second generation ofmonoclonal antibodies in which two different variable regions arecombined in the same molecule (Holliger 1999). Their use has beendemonstrated both in the diagnostic field and in the therapy field fromtheir capacity to recruit new effector functions or to target severalmolecules on the surface of tumor cells. Where bispecific antibodies areto be used, these may be conventional bispecific antibodies, which canbe manufactured in a variety of ways (Holliger 1993b), e.g. preparedchemically or from hybrid hybridomas, or may be any of the bispecificantibody fragments mentioned above. These antibodies can be obtained bychemical methods (Glennie 1987; Repp 1995) or somatic methods (Staerz1986; Suresh 1986) but likewise by genetic engineering techniques whichallow the heterodimerization to be forced and thus facilitate theprocess of purification of the antibody sought (Merchand 1998). Examplesof bispecific antibodies include those of the BiTe™ technology in whichthe binding domains of two antibodies with different specificity can beused and directly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against a target antigen, then a library can be made where theother arm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by alternative engineeringmethods as described in Ridgeway 1996.

Various methods are available in the art for obtaining antibodiesagainst a target antigen. The antibodies may be monoclonal antibodies,especially of human, murine, chimeric or humanized origin, which can beobtained according to the standard methods well known to the personskilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane 1988) or to the technique of preparationfrom hybridomas described by Kohler and Milstein, 1975.

Monoclonal antibodies can be obtained, for example, from an animal cellimmunized against A-FN or tenascin-C, or one of its fragments containingthe epitope recognized by said monoclonal antibodies, e.g. a fragmentcomprising or consisting of ED-A or the A1, A2, A3, A4, B or D domain ofthe tenascin-C large isoform, or a peptide fragment of ED-A or the A1,A2, A3, A4, B or D domain of the tenascin-C large isoform. The A-FN ortenascin-C, or one of their fragments, can especially be producedaccording to the usual working methods, by genetic recombinationstarting with a nucleic acid sequence contained in the cDNA sequencecoding for A-FN or tenascin-C, or fragment thereof, by peptide synthesisstarting from a sequence of amino acids comprised in the peptidesequence of the A-FN or tenascin-C and/or fragment thereof.

Monoclonal antibodies can, for example, be purified on an affinitycolumn on which A-FN or tenascin-C, or one of their fragments containingthe epitope recognized by said monoclonal antibodies, e.g. a fragmentcomprising or consisting of ED-A or tenascin-C, or a peptide fragment ofED-A or tenascin-C, has previously been immobilized. Monoclonalantibodies can be purified by chromatography on protein A and/or G,followed or not followed by ion-exchange chromatography aimed ateliminating the residual protein contaminants as well as the DNA and theLPS, in itself, followed or not followed by exclusion chromatography onSepharose gel in order to eliminate the potential aggregates due to thepresence of dimers or of other multimers. The whole of these techniquesmay be used simultaneously or successively.

Antigen-Binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that binds to and is complementary to all or part of thetarget antigen. Where an antigen is large, an antibody may only bind toa particular part of the antigen, which part is termed an epitope. Anantibody antigen-binding site may be provided by one or more antibodyvariable domains. An antibody antigen-binding site may comprise anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

Isolated

This refers to the state in which specific binding members for use inthe invention or nucleic acid encoding such specific binding members,will generally be in accordance with the present invention. Thus,specific binding members, VH and/or VL domains of the present inventionmay be provided isolated and/or purified, e.g. from their naturalenvironment, in substantially pure or homogeneous form, or, in the caseof nucleic acid, free or substantially free of nucleic acid or genes oforigin other than the sequence encoding a polypeptide with the requiredfunction. Isolated members and isolated nucleic acid will be free orsubstantially free of material with which they are naturally associatedsuch as other polypeptides or nucleic acids with which they are found intheir natural environment, or the environment in which they are prepared(e.g. cell culture) when such preparation is by recombinant DNAtechnology practised in vitro or in vivo. Specific binding members andnucleic acid may be formulated with diluents or adjuvants and still forpractical purposes be isolated—for example the members will normally bemixed with gelatin or other carriers if used to coat microtitre platesfor use in immunoassays, or will be mixed with pharmaceuticallyacceptable carriers or diluents when used in diagnosis or therapy.Specific binding members may be glycosylated, either naturally or bysystems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC85110503) cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

Heterogeneous preparations comprising antibody molecules may also beused in the invention. For example, such preparations may be mixtures ofantibodies with full-length heavy chains and heavy chains lacking theC-terminal lysine, with various degrees of glycosylation and/or withderivatized amino acids, such as cyclization of an N-terminal glutamicacid to form a pyroglutamic acid residue.

One or more specific binding members for an antigen, e.g. the A-FN, thetenascin-C, the ED-A of fibronectin, or the A1, A2, A3, A4, B or Ddomain of the tenascin-C large isoform may be obtained by bringing intocontact a library of specific binding members according to the inventionand the antigen or a fragment thereof, e.g. a fragment comprising orconsisting of ED-A or tenascin-C, or a peptide fragment of ED-A ortenascin-C and selecting one or more specific binding members of thelibrary able to bind the antigen.

An antibody library may be screened using Iterative Colony FilterScreening (ICFS). In ICFS, bacteria containing the DNA encoding severalbinding specificities are grown in a liquid medium and, once the stageof exponential growth has been reached, some billions of them aredistributed onto a growth support consisting of a suitably pre-treatedmembrane filter which is incubated until completely confluent bacterialcolonies appear. A second trap substrate consists of another membranefilter, pre-humidified and covered with the desired antigen.

The trap membrane filter is then placed onto a plate containing asuitable culture medium and covered with the growth filter with thesurface covered with bacterial colonies pointing upwards. The sandwichthus obtained is incubated at room temperature for about 16 h. It isthus possible to obtain the expression of the genes encoding antibodyfragments scFv having a spreading action, so that those fragmentsbinding specifically with the antigen which is present on the trapmembrane are trapped. The trap membrane is then treated to point outbound antibody fragments scFv with colorimetric techniques commonly usedto this purpose.

The position of the coloured spots on the trap filter allows to go backto the corresponding bacterial colonies which are present on the growthmembrane and produced the antibody fragments trapped. Such colonies aregathered and grown and the bacteria-a few millions of them aredistributed onto a new culture membrane repeating the proceduresdescribed above. Analogous cycles are then carried out until thepositive signals on the trap membrane correspond to single positivecolonies, each of which represents a potential source of monoclonalantibody fragments directed against the antigen used in the selection.ICFS is described in e.g. WO0246455, which is incorporated herein byreference. A library may also be displayed on particles or molecularcomplexes, e.g. replicable genetic packages such bacteriophage (e.g. T7)particles, or other in vitro display systems, each particle or molecularcomplex containing nucleic acid encoding the antibody VH variable domaindisplayed on it, and optionally also a displayed VL domain if present.Phage display is described in WO92/01047 and e.g. U.S. Pat. No.5,969,108, U.S. Pat. No. 5,565,332, U.S. Pat. No. 5,733,743, U.S. Pat.No. 5,858,657, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,872,215, U.S.Pat. No. 5,885,793, U.S. Pat. No. 5,962,255, U.S. Pat. No. 6,140,471,U.S. Pat. No. 6,172,197, U.S. Pat. No. 6,225,447, U.S. Pat. No.6,291,650, U.S. Pat. No. 6,492,160 and U.S. Pat. No. 6,521,404, each ofwhich is herein incorporated by reference in its entirety.

Following selection of binding members able to bind the antigen anddisplayed on bacteriophage or other library particles or molecularcomplexes, nucleic acid may be taken from a bacteriophage or otherparticle or molecular complex displaying a said selected binding member.Such nucleic acid may be used in subsequent production of a bindingmember or an antibody VH or VL variable domain by expression fromnucleic acid with the sequence of nucleic acid taken from abacteriophage or other particle or molecular complex displaying a saidselected binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected binding member may beprovided in isolated form, as may a binding member comprising such a VHdomain.

Ability to bind the A-FN or the tenascin-C, or the ED-A of fibronectinor the A1, A2, A3, A4, B or D domain of the tenascin-C large isoform, orother target antigen or isoform may be further tested, e.g. ability tocompete with e.g. any one of anti-ED-A antibodies H1, B2, C5, D5, E5,C8, F8, F1, B7, E8 or G9 for binding to the A-FN or a fragment of theA-FN, e.g. the ED-A of fibronectin, or an anti-tenascin-C antibody, suchas F16 or 4A1-F16, for binding to the tenascin-C or a fragment oftenascin-C, e.g. the A1, A2, A3, A4, B or D domain of the tenascin-Clarge isoform.

A specific binding member for use in the invention may bind the A-FNand/or the ED-A of fibronectin, or tenascin-C and/or the A1, A2, A3, A4,B or D domain of the tenascin-C isoform specifically. A specific bindingmember of the present invention may bind the A-FN and/or the ED-A offibronectin, or the tenascin-C, and/or the A1 domain of the tenascin-Clarge isoform, with the same affinity as an anti-ED-A antibody H1, B2,C5, D5, E5, C8, F8, F1, B7, E8 or G9, e.g. in scFv format, or as theanti-tenascin-C antibodies F16 or 4A1-F16, respectively, or with anaffinity that is better. A specific binding member for use in theinvention may bind the A-FN and/or the ED-A of fibronectin, or thetenascin-C and/or the A1, A2, A3, A4, B or D domain of the tenascin-Clarge isoform, with a K_(D) of 3×10⁻⁸ M or an affinity that is better.Preferably, a specific binding member for use in the invention binds theA-FN and/or the ED-A of fibronectin, or the tenascin-C and/or the A1,A2, A3, A4, B or D domain of the tenascin-C large isoform, with a K_(D)of 2×10⁻⁸ M or an affinity that is better. More preferably, a specificbinding member for use in the invention binds the A-FN and/or the ED-Aof fibronectin, or the tenascin-C and/or the A1, A2, A3, A4, B or Ddomain of the tenascin-C large isoform, with a K_(D) of 1.7×10⁻⁸ M or anaffinity that is better. Yet more preferably, a specific binding memberfor use in the invention binds the A-FN and/or the ED-A of fibronectin,or the tenascin-C and/or the A1, A2, A3, A4, B or D domain of thetenascin-C large isoform, with a K_(D) of 1.4×10⁻⁸ M or an affinity thatis better. Most preferably, a specific binding member for use in theinvention binds the A-FN and/or the ED-A of fibronectin, or thetenascin-C and/or the A1, A2, A3, A4, B or D domain of the tenascin-Clarge isoform, with a K_(D) of 3×10⁻⁹ M or an affinity that is better.

A specific binding member of the present invention may bind to the sameepitope on A-FN and/or the ED-A of fibronectin as one of the anti-ED-Aantibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9, or to the sameepitope on tenascin-C, and/or the A1 domain of the tenascin-C largeisoform, as the anti-tenascin-C antibody F16 or 4A1-F16.

A specific binding member for use in the invention may not show anysignificant binding to molecules other than to the A-FN and/or the ED-Aof fibronectin, or to the tenascin-C and/or the A1, A2, A3, A4, B or Ddomain of the tenascin-C large isoform. In particular, the specificbinding member may not bind other isoforms of fibronectin, for examplethe ED-B isoform and/or the IIICS isoform of fibronectin, or otherisoforms of tenascin-C, for example the tenascin-C small isoform.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. The techniques required to make substitutionswithin amino acid sequences of CDRs, antibody VH or VL domains andbinding members generally are available in the art. Variant sequencesmay be made, with substitutions that may or may not be predicted to havea minimal or beneficial effect on activity, and tested for ability tobind A-FN and/or the ED-A of fibronectin, or tenascin-C and/or the A1,A2, A3, A4, B or D domain of the tenascin-C large isoform, and/or forany other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, may be 5, 4, 3, 2 or 1. Alterations may be made in one ormore framework regions and/or one or more CDRs. The alterations normallydo not result in loss of function, so a specific binding membercomprising a thus-altered amino acid sequence may retain an ability tobind A-FN and/or the ED-A of fibronectin, or tenascin-C and/or the A1,A2, A3, A4, B or D domain of the tenascin-C large isoform. For example,it may retain the same quantitative binding as a specific binding memberin which the alteration is not made, e.g. as measured in an assaydescribed herein. The specific binding member comprising a thus-alteredamino acid sequence may have an improved ability to bind A-FN and/or theED-A of fibronectin, or tenascin-C and/or the A1, A2, A3, A4, B or Ddomain of the tenascin-C large isoform.

Novel VH or VL regions carrying CDR-derived sequences for use in theinvention may be generated using random mutagenesis of one or moreselected VH and/or VL genes to generate mutations within the entirevariable domain. In some embodiments one or two amino acid substitutionsare made within an entire variable domain or set of CDRs. Another methodthat may be used is to direct mutagenesis to CDR regions of VH or VLgenes.

As noted above, a CDR amino acid sequence substantially as set outherein may be carried as a CDR in a human antibody variable domain or asubstantial portion thereof. The HCDR3 sequences substantially as setout herein represent embodiments of the present invention and forexample each of these may be carried as a HCDR3 in a human heavy chainvariable domain or a substantial portion thereof.

Variable domains employed in the invention may be obtained or derivedfrom any germ-line or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A variable domain can be derived from anon-human antibody. A CDR sequence for use in the invention (e.g. CDR3)may be introduced into a repertoire of variable domains lacking a CDR(e.g. CDR3), using recombinant DNA technology. For example, Marks et al.(1992) describe methods of producing repertoires of antibody variabledomains in which consensus primers directed at or adjacent to the 5′ endof the variable domain area are used in conjunction with consensusprimers to the third framework region of human VH genes to provide arepertoire of VH variable domains lacking a CDR3. Marks et al. furtherdescribe how this repertoire may be combined with a CDR3 of a particularantibody. Using analogous techniques, the CDR3-derived sequences of thepresent invention may be shuffled with repertoires of VH or VL domainslacking a CDR3, and the shuffled complete VH or VL domains combined witha cognate VL or VH domain to provide binding members for use in theinvention. The repertoire may then be displayed in a suitable hostsystem such as the phage display system of WO92/01047, which is hereinincorporated by reference in its entirety, or any of a subsequent largebody of literature, including Kay, Winter & McCafferty (1996), so thatsuitable binding members may be selected. A repertoire may consist offrom anything from 10⁴ individual members upwards, for example at least10⁹, at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹ or at least10¹⁰ members.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a bindingmember or binding members for the A-FN and/or the ED-A of fibronectin,or the tenascin-C and/or the A1, A2, A3, A4, B or D domain of thetenascin-C large isoform.

One or more of the HCDR1, HCDR2 and HCDR3 of antibody H1, B2, C5, D5,E5, C8, F8, F1, B7, E8, G9, F16, or 4A1-F16, or the set of HCDRs ofantibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8, G9, F16 or 4A1-F16 maybe employed, and/or one or more of the LCDR1, LCDR2 and LCDR3 ofantibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8, G9, F16, or 4A1-F16, orthe set of LCDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8, G9,F16 or 4A1-F16 may be employed.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRsand/or sets of LCDRs disclosed herein may be employed.

The A-FN and/or the ED-A of fibronectin, or the tenascin-C and/or theA1, A2, A3, A4, B or D domain of the tenascin-C large isoform, may beused in a screen for specific binding members, e.g. antibody molecules,for use in the preparation of a medicament for the treatment ofendometriosis, psoriasis, or psoriatic arthritis. The screen may ascreen of a repertoire as disclosed elsewhere herein.

A substantial portion of an immunoglobulin variable domain may compriseat least the three CDR regions, together with their interveningframework regions. The portion may also include at least about 50% ofeither or both of the first and fourth framework regions, the 50% beingthe C-terminal 50% of the first framework region and the N-terminal 50%of the fourth framework region. Additional residues at the N-terminal orC-terminal end of the substantial part of the variable domain may bethose not normally associated with naturally occurring variable domainregions. For example, construction of specific binding members of thepresent invention made by recombinant DNA techniques may result in theintroduction of N- or C-terminal residues encoded by linkers introducedto facilitate cloning or other manipulation steps. Other manipulationsteps include the introduction of linkers to join variable domainsdisclosed elsewhere herein to further protein sequences includingantibody constant regions, other variable domains (for example in theproduction of diabodies) or detectable/functional labels as discussed inmore detail elsewhere herein.

Although specific binding members may comprise a pair of VH and VLdomains, single binding domains based on either VH or VL domainsequences may also be used in the invention. It is known that singleimmunoglobulin domains, especially VH domains, are capable of bindingtarget antigens in a specific manner. For example, see the discussion ofdAbs above.

In the case of either of the single binding domains, these domains maybe used to screen for complementary domains capable of forming atwo-domain binding member able to bind A-FN and/or the ED-A offibronectin, or tenascin-C, and/or the A1, A2, A3, A4, B or D domain ofthe tenascin-C large isoform. This may be achieved by phage displayscreening methods using the so-called hierarchical dual combinatorialapproach as disclosed in WO92/01047, herein incorporated by reference inits entirety, in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain binding member isselected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Marks1992.

Specific binding members for use in the present invention may furthercomprise antibody constant regions or parts thereof, e.g. human antibodyconstant regions or parts thereof. For example, a VL domain may beattached at its C-terminal end to antibody light chain constant domainsincluding human Cκ or Cλ chains, e.g. Cλ. Similarly, a specific bindingmember based on a VH domain may be attached at its C-terminal end to allor part (e.g. a CH1 domain) of an immunoglobulin heavy chain derivedfrom any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of theisotype sub-classes, particularly IgG1 and IgG4. Any synthetic or otherconstant region variant that has these properties and stabilizesvariable regions is also useful in embodiments of the present invention.

Specific binding members for use in the invention may be labelled with adetectable or functional label. A label can be any molecule thatproduces or can be induced to produce a signal, including but notlimited to fluorescers, radiolabels, enzymes, chemiluminescers orphotosensitizers. Thus, binding may be detected and/or measured bydetecting fluorescence or luminescence, radioactivity, enzyme activityor light absorbance. Detectable labels may be attached to antibodies foruse in the invention using conventional chemistry known in the art.

There are numerous methods by which the label can produce a signaldetectable by external means, for example, by visual examination,electromagnetic radiation, heat, and chemical reagents. The label canalso be bound to another specific binding member that binds the antibodyfor use in the invention, or to a support.

Labelled specific binding members, e.g. scFv labelled with a detectablelabel, may be used diagnostically in vivo, ex vivo or in vitro, and/ortherapeutically.

For example, radiolabelled binding members (e.g. binding membersconjugated to a radioisotope) may be used in radiodiagnosis andradiotherapy. Radioisotopes which may be conjugated to a binding memberfor use in the invention include isotopes such as ^(94m)Tc, ^(99m)Tc,¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ¹¹¹In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁸⁶Y,⁸⁸Y, ⁹⁰Y, ¹²¹Sn, ¹⁶¹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁰⁵Rh, ¹⁷⁷Lu, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁸F, ²¹¹At and ²²⁵Ac. Preferably, positron emitters, such as ¹⁸Fand ¹²⁴I, or gamma emitters, such as ^(99m)Tc, ¹¹¹In and ¹²³I, are usedfor diagnostic applications (e.g. for PET), while beta-emitters, such as¹³¹I, ⁹⁰Y and ¹⁷⁷Lu, are preferably used for therapeutic applications.Alpha-emitters, such as ²¹¹At and ²²⁵Ac may also be used for therapy.

For example, a specific binding member for use in the invention labelledwith a detectable label may be used to detect, diagnose or monitorendometriosis, psoriasis, or psoriatic arthritis in a human or animal.

A specific binding member of the present invention may be used for themanufacture of a diagnostic product for use in diagnosing endometriosis,psoriasis, or psoriatic arthritis.

The present invention provides a method of detecting or diagnosingendometriosis, psoriasis, or psoriatic arthritis in a human or animalcomprising:

-   -   (a) administering to the human or animal a specific binding        member of the present invention, for example labelled with a        detectable label, which binds the ED-A isoform of fibronectin        and/or the ED-A of fibronectin, or tenascin-C and/or the A1, A2,        A3, A4, B and/or D domain of the tenascin-C large isoform, and    -   (b) determining the presence or absence of the specific binding        member in sites of endometriosis, psoriasis, or psoriatic        arthritis of the human or animal body;        wherein localisation of the specific binding member to sites of        endometriosis, psoriasis, or psoriatic arthritis indicates the        presence of endometriosis, psoriasis, or psoriatic arthritis.

Where the binding member is labelled with a detectable label, thepresence or absence of the detectable label may be determined bydetecting the label.

A conjugate or fusion between a binding member for use in the inventionand a molecule that exerts a biocidal, cytotoxic immunosuppressive oranti-inflammatory effect on target cells in the lesions and an antibodydirected against an extracellular matrix component which is present insuch lesions may be employed in the present invention. For example, theconjugated molecule may be inter alia interleukin-10, TGF-β, IL-2,IL-12, IL-15, IL-21, IL-24, IL-33, tumour necrosis factor (TNF), orinterferon-α,-β or -γ, an anti-inflammatory or other drug, aphotosensitizer or a radionuclide. Such conjugates may be usedtherapeutically, e.g. for treatment of endometriosis, psoriasis, orpsoriatic arthritis as referred to herein.

Production and use of fusions or conjugates of specific binding memberswith biocidal or cytotoxic molecules is described for example inWO01/62298, which is incorporated by reference herein.

The invention provides a method of treating endometriosis, psoriasis, orpsoriatic arthritis, the method comprising administering to anindividual a therapeutically effective amount of a medicament comprisinga specific binding member for use in the invention.

The specific binding member for use in the invention may be a conjugateof (i) a molecule which exerts an anti-inflammatory effect on targetcells by cellular interaction, an anti-inflammatory molecule, a cytokinee.g. IL-10, TGF-β, IL-2, IL-12, IL-15, IL-21, IL-24, IL-33, tumournecrosis factor (TNF), interferon-α, -β or -γ, or other drug, and (ii) aspecific binding member for the ED-A isoform of fibronectin and/or theED-A of fibronectin, or tenascin-C, and/or the A1, A2, A3, A4, B and/orD domain of the tenascin-C large isoform.

The specific binding member for use in the invention may be a conjugateof (i) a molecule which exerts an immunosuppressive or anti-inflammatoryeffect and (ii) a specific binding member for the ED-A isoform offibronectin and/or the ED-A of fibronectin, or tenascin-C, and/or theA1, A2, A3, A4, B and/or D domain of the tenascin-C large isoform.

The specific binding member for use in the invention may be a conjugateof (i) interleukin-10 (IL10) or TGF beta and (ii) a specific bindingmember for the ED-A isoform of fibronectin and/or the ED-A offibronectin, or tenascin-C, and/or the A1, A2, A3, A4, B and/or D domainof the tenascin-C large isoform. Such a specific binding member isuseful in aspects of the invention disclosed herein relating totreatment of endometriosis, psoriasis and psoriatic arthritis.

The invention provides the use of a specific binding member as describedherein for the preparation of a medicament for the treatment ofendometriosis, psoriasis and psoriatic arthritis.

The specific binding member for use in the invention may be a conjugatedor fused to a molecule that exerts a biocidal, cytotoxic,immunosuppressive or anti-inflammatory effect as described herein. Thespecific binding member for use in the invention may be a conjugate of(i) a molecule which exerts a biocidal or cytotoxic effect on targetcells by cellular interaction or has an immunosuppressive oranti-inflammatory effect and (ii) a specific binding member for the ED-Aisoform of fibronectin and/or the ED-A of fibronectin, or tenascin-C,and/or the A1, A2, A3, A4, B and/or D domain of the tenascin-C largeisoform.

Also described herein is a conjugate of (i) a molecule which exerts abiocidal or cytotoxic effect on target cells by cellular interaction, oran immunosuppressive or anti-inflammatory effect and (ii) a bindingmember for the ED-A isoform of fibronectin and/or the ED-A offibronectin, or tenascin-C, and/or the A1, A2, A3, A4, B and/or D domainof the tenascin-C large isoform. Such a conjugate preferably comprises afusion protein comprising the biocidal, cytotoxic, immunosuppressive oranti-inflammatory molecule and a said binding member, or, where thebinding member is two-chain or multi-chain, a fusion protein comprisingthe biocidal, cytotoxic, immunosuppressive or anti-inflammatory moleculeand a polypeptide chain component of said binding member. Preferably thebinding member is a single-chain polypeptide, e.g. a single-chainantibody molecule, such as scFv.

A fusion protein comprising the immunosuppressive or anti-inflammatorymolecule and a single-chain Fv antibody molecule may be used in theinvention.

The immunosuppressive or anti-inflammatory molecule that exerts itseffect on target cells by cellular interaction, may interact directlywith the target cells, may interact with a membrane-bound receptor onthe target cell or perturb the electrochemical potential of the cellmembrane. Preferably, the molecule is IL-10 or TGF-β.

Examples of other molecules which can be conjugated to the specificbinding member include IL-2, IL-12, IL-15, IL-21, IL-24, IL-33, tumournecrosis factor (TNF), or interferon-α, -β or -γ.

As discussed further below, the specific binding member for use in theinvention is preferably an antibody molecule or comprises an antibodyantigen-binding site. Conveniently, the specific binding member may be asingle-chain polypeptide, such as a single-chain antibody. This allowsfor convenient production of a fusion protein comprising single-chainantibody and, for example, immunosuppressive or anti-inflammatorymolecule (e.g. interleukin-10 or TGF beta). An antibody antigen-bindingsite may be provided by means of association of an antibody VH domainand an antibody VL domain in separate polypeptides, e.g. in a completeantibody or in an antibody fragment such as Fab or diabody. Where thespecific binding member is a two-chain or multi-chain molecule (e.g. Fabor whole antibody, respectively), an immunosuppressive oranti-inflammatory molecule, for example, may be conjugated as a fusionpolypeptide with one or more polypeptide chains in the specific bindingmember.

The specific binding member may be conjugated with the immunosuppressiveor anti-inflammatory molecule by means of a peptide bond, i.e. within afusion polypeptide comprising said molecule and the specific bindingmember or a polypeptide chain component thereof (see e.g. Trachsel etal.). Other means for conjugation include chemical conjugation,especially cross-linking using a bifunctional reagent (e.g. employingDOUBLE-REAGENTS™ Cross-linking Reagents Selection Guide, Pierce).

Also described herein is isolated nucleic acid encoding a specificbinding member for use in the present invention. Nucleic acid mayinclude DNA and/or RNA. A nucleic acid may code for a CDR or set of CDRsor VH domain or VL domain or antibody antigen-binding site or antibodymolecule, e.g. scFv or IgG, e.g. IgG1, as defined above. The nucleotidesequences may encode the VH and/or VL domains disclosed herein.

Further described herein are constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as described above.

A recombinant host cell that comprises one or more constructs as aboveare also described. A nucleic acid encoding any CDR or set of CDRs or VHdomain or VL domain or antibody antigen-binding site or antibodymolecule, e.g. scFv or IgG1 or IgG4 as provided, is described, as is amethod of production of the encoded product, which method comprisesexpression from encoding nucleic acid. Expression may conveniently beachieved by culturing under appropriate conditions recombinant hostcells containing the nucleic acid. Following production by expression aVH or VL domain, or specific binding member may be isolated and/orpurified using any suitable technique, then used as appropriate.

A nucleic acid may comprise DNA or RNA and may be wholly or partiallysynthetic. Reference to a nucleotide sequence as set out hereinencompasses a DNA molecule with the specified sequence, and encompassesa RNA molecule with the specified sequence in which U is substituted forT, unless context requires otherwise.

A method of production of an antibody VH variable domain, the methodincluding causing expression from encoding nucleic acid is alsodescribed. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals. The expression ofantibodies and antibody fragments in prokaryotic cells is wellestablished in the art. For a review, see for example Plückthun 1991. Acommon bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a specific bindingmember for example Chadd & Chamow (2001), Andersen & Krummen (2002),Larrick & Thomas (2001). Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanomacells, YB2/0 rat myeloma cells, human embryonic kidney cells, humanembryonic retina cells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. ‘phage, as appropriate. For further details see,for example, Sambrook & Russell (2001). Many known techniques andprotocols for manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in Ausubel 1999.

A host cell may contain a nucleic acid as described herein. Such a hostcell may be in vitro and may be in culture. Such a host cell may be invivo. In vivo presence of the host cell may allow intracellularexpression of a binding member for use in the present invention as“intrabodies” or intracellular antibodies. Intrabodies may be used forgene therapy.

A method comprising introducing a nucleic acid disclosed herein into ahost cell is also described. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay be incorporated into the host cell or into an artificial chromosome.Incorporation may be either by random or targeted integration of one ormore copies at single or multiple loci. For bacterial cells, suitabletechniques may include calcium chloride transformation, electroporationand transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene. The purification of the expressed product may beachieved by methods known to one of skill in the art.

The nucleic acid may be integrated into the genome (e.g. chromosome) ofthe host cell. Integration may be promoted by inclusion of sequencesthat promote recombination with the genome, in accordance with standardtechniques.

A method that comprises using a construct as stated above in anexpression system in order to express a specific binding member orpolypeptide as above is also described.

Specific binding members for use in the present invention are designedto be used in methods of diagnosis or treatment in human or animalsubjects, e.g. human. Specific binding members for use in the inventionmay be used in diagnosis or treatment of endometriosis, psoriasis andpsoriatic arthritis.

Accordingly, the invention provides methods of treatment comprisingadministration of a specific binding member as described, pharmaceuticalcompositions comprising such a specific binding member, and use of sucha specific binding member in the manufacture of a medicament foradministration, for example in a method of making a medicament orpharmaceutical composition comprising formulating the specific bindingmember with a pharmaceutically acceptable excipient. Pharmaceuticallyacceptable vehicles are well known and will be adapted by the personskilled in the art as a function of the nature and of the mode ofadministration of the active compound(s) chosen.

Specific binding members for use in the present invention will usuallybe administered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember. Thus, pharmaceutical compositions described herein, and for usein accordance with the present invention, may comprise, in addition toactive ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, inhaled or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration such as for examplenanobodies etc are also envisaged in the present invention. Such oralformulations may be in tablet, capsule, powder, liquid or semi-solidform. A tablet may comprise a solid carrier such as gelatin or anadjuvant. Liquid pharmaceutical compositions generally comprise a liquidcarrier such as water, petroleum, animal or vegetable oils, mineral oilor synthetic oil. Physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol may be included.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be employed, as required. Many methods for thepreparation of pharmaceutical formulations are known to those skilled inthe art. See e.g. Robinson, 1978.

A composition may be administered alone or in combination with othertreatments, concurrently or sequentially or as a combined preparationwith another therapeutic agent or agents, dependent upon the conditionto be treated.

A specific binding member for use in the present invention may be usedas part of a combination therapy in conjunction with an additionalmedicinal component. Combination treatments may be used to providesignificant synergistic effects, particularly the combination of aspecific binding member for use in the present invention with one ormore other drugs. A specific binding member for use in the presentinvention may be administered concurrently or sequentially or as acombined preparation with another therapeutic agent or agents, for thetreatment of one or more of the conditions listed herein.

For example, a specific binding member for use in the invention may beused in combination with an existing therapeutic agent for the treatmentof endometriosis, psoriasis or psoriatic arthritis.

A specific binding member for use in the invention and one or more ofthe above additional medicinal components may be used in the manufactureof a medicament. The medicament may be for separate or combinedadministration to an individual, and accordingly may comprise thespecific binding member and the additional component as a combinedpreparation or as separate preparations. Separate preparations may beused to facilitate separate and sequential or simultaneousadministration, and allow administration of the components by differentroutes e.g. oral and parenteral administration.

In accordance with the present invention, compositions provided may beadministered to mammals. Administration may be in a “therapeuticallyeffective amount”, this being sufficient to show benefit to a patient.Such benefit may be at least amelioration of at least one symptom. Thus“treatment of endometriosis, psoriasis or psoriatic arthritis” refers toamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the composition, the type ofspecific binding member, the method of administration, the scheduling ofadministration and other factors known to medical practitioners.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andmay depend on the severity of the symptoms and/or progression of adisease being treated. Appropriate doses of antibody are well known inthe art (Ledermann 1991 and Bagshawe 1991. Specific dosages indicatedherein, or in the Physician's Desk Reference (2003) as appropriate forthe type of medicament being administered, may be used. Atherapeutically effective amount or suitable dose of a specific bindingmember for use in the invention can be determined by comparing its invitro activity and in vivo activity in an animal model. Methods forextrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for diagnosis, prevention or fortreatment, the size and location of the area to be treated, the precisenature of the antibody (e.g. whole antibody, fragment or diabody), andthe nature of any detectable label or other molecule attached to theantibody. A typical antibody dose will be in the range 100 μg to 1 g forsystemic applications, and 1 μg to 1 mg for topical applications. Aninitial higher loading dose, followed by one or more lower doses, may beadministered. An antibody may be a whole antibody, e.g. the IgG1 or IgG4isotype. This is a dose for a single treatment of an adult patient,which may be proportionally adjusted for children and infants, and alsoadjusted for other antibody formats in proportion to molecular weight.Treatments may be repeated at daily, twice-weekly, weekly or monthlyintervals, at the discretion of, the physician. Treatments may be everytwo to four weeks for subcutaneous administration and every four toeight weeks for intravenous administration. In some embodiments of thepresent invention, treatment is periodic, and the period betweenadministrations is about two weeks or more, e.g. about three weeks ormore, about four weeks or more, or about once a month. In otherembodiments of the invention, treatment may be given before, and/orafter surgery, and may be administered or applied directly at theanatomical site of surgical treatment.

Further aspects and embodiments of the invention will be apparent tothose skilled in the art given the present disclosure including thefollowing experimental exemplification.

EXPERIMENTAL Results Histochemical Analysis of Human EndometrioticSpecimens

Expression of fibronectin domains ED-A and ED-B and the A1 domain of thetenascin-C large isoform was investigated by immunohistochemistry onhuman endometriotic specimens using biotinylated F8-, L19- and F16-SIPantibodies respectively. The results of the immunohistochemical analysisare shown in FIG. 1.

In FIG. 1 darker staining indicates expression of the respectiveantigens (indicated with black arrows).

Both the ED-A of fibronectin (recognised by the biotinylated F8-SIPantibody) and the A1 domain of tenascin-C (recognised by thebiotinylated F16-SIP antibody) were strongly expressed around theperivascular structures of biopsies of human endometriotic specimens(see FIGS. 1 A,B&C for staining with F8-SIP and FIGS. 1 G,H&I forstaining with F16-SIP). The intense staining of vascular structures withF16-SIP was stronger than observed with F8-SIP. No staining is visiblefor the negative control, i.e. the same type of endometriotic specimenincubated with the streptavidin reagent, but without any primaryantibody (see FIGS. 1 J,K&L).

The ED-B domain of fibronectin (recognised by the biotinylated L19-SIPantibody) was only weakly positive (see FIGS. 1 D,E&F).

Histochemical Analysis of Human Ulcerative Colitic Specimens

Expression of fibronectin domains ED-A and ED-B, and the A1 domain ofthe tenascin-C large isoform were investigated by immunohistochemistryon human ulcerative colitic specimens using biotinylated F8-, L19- andF16-SIP antibodies respectively. The results of the immunohistochemicalanalysis are shown in FIG. 2.

In FIG. 2, areas of negative staining around perivascular structures areindicated using white arrows.

Immunohistochemical analysis of human ulcerative colitic samplesrevealed virtually negative staining for all three antibodies, F8, L19and F16 (see FIGS. 2A,B&C respectively). Only a weak positivity wasobserved with F8-SIP in some specimens.

Histochemical Analysis of Human Psoriatic Arthritic Specimens

Expression of fibronectin domains ED-A and ED-B, and the A1 domain ofthe tenascin-C large isoform were investigated by immunohistochemistryon human psoriatic arthritic specimens using biotinylated F8-, L19- andF16-SIP antibodies respectively. The results of the immunohistochemicalanalysis are shown in FIG. 3.

In FIG. 3, darker staining indicates expression of the respectiveantigens (indicated with black arrows).

The A1 domain of tenascin-C (recognised by the biotinylated F16-SIPantibody) was strongly expressed around the perivascular structures ofbiopsies of human psoriatic arthritic specimens, i.e. very intensestaining was observed (see FIG. 3C). No staining was visible for thenegative control nor for the ED-B domain of fibronectin (recognised bythe biotinylated L19-SIP antibody) or the ED-A domain of fibronectin(recognised by the biotinylated F8-SIP antibody) (see FIGS. 3 A, B and Drespectively).

Histochemical Analysis of Samples from Patients with Multiple Sclerosis

Expression of fibronectin domains ED-A and ED-B, and the A1 domain ofthe tenascin-C large isoform were investigated by immunohistochemistryon samples from patients with multiple sclerosis using biotinylated F8-,L19- and F16-SIP antibodies respectively. The results of theimmunohistochemical analysis are shown in FIG. 4.

In FIG. 4, areas of negative staining around perivascular structures areindicated using white arrows.

Immunohistochemical analysis of tissue sections from pathologicalspecimens from patients with multiple sclerosis revealed only extremelyweak positivity at vascular structures for L19, F8 and F16 (see FIGS. 4A,B&C).

Histochemical Analysis of Samples from Patients with Psoriasis

Expression of fibronectin domains ED-A and ED-B, and the A1 domain ofthe tenascin-C large isoform were investigated by immunohistochemistryon samples from patients with psoriasis using biotinylated F8-, L19- andF16-SIP antibodies respectively. The results of the immunohistochemicalanalysis are shown in FIG. 5.

In FIG. 5, darker staining indicates expression of the respectiveantigens (indicated with black arrows).

The A1 domain of tenascin-C (recognised by the biotinylated F16-SIPantibody) was strongly expressed around the perivascular structures ofbiopsies of human psoriatic specimens, i.e. very intense staining wasobserved (see FIG. 5C). No staining was visible for the negative controlnor for the ED-B domain of fibronectin (recognised by the biotinylatedL19-SIP antibody) or the ED-A domain of fibronectin (recognised by thebiotinylated F8-SIP antibody) (see FIGS. 5 D, B and A respectively).

Based on the findings described above, expression of ED-A and thetenascin-C large isoform is not found in all angiogenesis-relateddiseases. Endometriosis is particularly suited for pharmacodeliveryusing anti-ED-A antibody molecules, while endometriosis, psoriasis andpsoriatic arthritis are particularly suited for pharmacodelivery usinganti-tenascin-C antibody molecules.

Near Infrared Imaging of Endometriotic Lesions in Mice

A mouse model of endometriosis was generated as described below. Theselective accumulation of F8-SIP in mice with endometriosis was testedby near infrared imaging analysis, as described by Birchler et al.[Birchler et al., J Immunological Methods 1999, 231, 239-248]. F8-SIPand F16-SIP were labeled using Alexa750 (Molecular Probes), according tothe manufacturer's recommendations, and injected into the tail vein ofendometriosis mice. Mice were sacrificed and imaged in a near infraredmouse imager 24 hours after injection. F16-SIP was used as a negativecontrol as it recognises the human A1 domain of tenascin-C but not themurine antigen.

As shown in FIGS. 10 A and B, SIP(F8)-ALEXA750 accumulates on theendometriotic lesions (indicated by arrows), whereas SIP(F16)-ALEXA750does not (FIG. 10C), thus confirming the in vivo specificity of F8-SIP.

Ex Vivo Detection of SIP(F8)-ALEXA750

As soon as the near infrared imaging was completed, endometrioticlesions were removed from sacrificed animals, embedded in cryoembeddingcompound (Microm, Walldorf, Germany) and stored at −80° C. Sections (10μm) were then cut and fixed in acetone. SIP(F8)-ALEXA750 was detectedusing a rabbit anti-human IgE antibody (Dako, Glostrup, Denmark),followed by Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes,Leiden, The Netherlands). For the detection of blood vessels, doublestaining with a rat anti-CD31 antibody followed by Alexa Fluor 594donkey anti-rat IgG was performed. As a negative control, the specimenwas probed with Alexa Fluor 488 goat anti-rabbit IgG minus the human IgEantibody.

The results clearly show correspondence between the blood vessels (seebrighter areas in FIGS. 11 D, E and F) and staining withSIP(F8)-ALEXA750 (see brighter areas in FIGS. 11 A, B and C). Theseresults confirm that SIP(F8)-ALEXA750 had accumulated around vascularstructures during the imaging studies.

These data show the first successful attempt at in vivo imaging ofendometriosis without the need for laparoscopy, or other form ofsurgery.

Materials and Methods

Immunohistochemistry with Biotinylated SIP Antibodies

The tissue sections were fixed in cold acetone (−20° C.) for 10 minutesand the slides were dried at room temperature for 30 minutes. Siliconwas applied using a pen and the slides were then immersed in TBS (50 mMTris, 100 mM NaCl, pH 7.4, 0.01% aprotinin) for 5 minutes. The slideswere dried with paper without touching the sections. The sections wereblocked with 20% fetal calf serum (FCS) in TBS for 30 minutes. Theblocking solution was then removed and the slides were submerged in TBSfor 5 minutes. The primary biotinylated antibody in SIP format wasdiluted in TBS/3% BSA to a final concentration of 1.6 μg/ml and appliedto the sections for 60 minutes at room temperature. The slides werewashed twice with TBS+2 mM MgCl₂ (406 mg/1) (2×5 minutes). The back ofthe slides were dried with paper and SAP-complex 1:150 (Biopsa F014-62)in TBS+2 mM MgCl₂/3% BSA for 60 minutes at room temperature. Thesections were washed three times with TBS+2 mM MgCl₂ (3×5 minutes). Thesubstrate (made up by dissolving 1 Tris and 1 FastRed tablet per 1 mlMilliQ water) was added and incubated on the sections for 10 minutes.The sections were washed twice with deionised water (2×2 minutes) andtransferred to Gill's hematoxylin solution no. 2 for 2 minutes. Theslides were quickly transferred to de-ionised water and rinsed withwater for 5 minutes. The slides were allowed to dry and mounted withglycerol and visualised with an optical microscope (Zeiss Axiovert 5100TV).

Mouse Model of Endometriosis

6-8 week old C57BL/6 mice were subjected to ovariectomy 7 days prior toinduction of endometriosis. Mice were anesthesized by isoflurane incombination with carprofen (Rimadyl). After ovariectomy, mice wereoestrogen-treated (3×/week 4ug estradiol/mouse in a volume of 100 ularachis oil subcutanously injected, starting at the day oftransplantation). Ovariectomy plus oestrogen supplementation was done inorder to abrogate differences related to the stage of the oestrouscycle. At day 0, mice were split into 2 groups: donor mice (33%) andrecipient mice (66%). Donor mice were killed and both uterine horns wereremoved and subsequently placed in a sterile Petri dish containingsterile saline. Endometrium was detached from the uterine muscle andfinley chopped using a scalpel. Endometrial fragments were suspended insaline and injected into the peritoneal cavity of the receipient mice. 4weeks after transplantation, mice were used for near infrared imaging.

TABLE 1 Nucleotide sequences of the heavy chains (VH) of anti-ED-A andanti-tenascin C antibodies. VH CDR1 sequences are underlined; VHCDR2 sequences are in italics and underlined; VH CDR 3 sequencesare in bold and underlined. Antibody VH domain H1GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCGCGGAGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT AGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 1) B2GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGCGGCTAAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT AGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 3) C5GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCGATTACTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 5) D5GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGTGATGAAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT AGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 7) E5GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCICTGGATTCACCTTTAGCACTGGTTCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 9) C8GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTTCAGACTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 11) F8 VHGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 13) F8 VH V5LGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 15) F1GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTAGGCGCGTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT AGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 17) B7GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCATTTTGATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 19) E8GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGATATGCATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 21) G9GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCATATGCAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATT A GTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA AGTACTCATTTGTAT CTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 23) F16GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGGTATGGTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA GCGCATAATGCTTTT GACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCGAGA (SEQ ID NO: 25) 4A1-F16GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGGTATGGTGCGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAA GCGCATAATGCTTTT GACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCGAGA (SEQ ID NO: 27)

TABLE 2 Amino acid sequences of the heavy chains (VH) of anti-ED-A andanti-tenascin C antibodies. VH CDR1 sequences are underlined; VHCDR2 sequences are in italics and underlined; VH CDR 3 sequencesare in bold and underlined. Antibody VH domain H1EVQLVESGGGLVQPGGSLRLSCAASGFTFSPRRMSWVRQAPGKGLEWVSAI SG SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 2) B2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAAKMSWVRQAPGKGLEWVSAI SGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 4) C5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSPITMSWVRQAPGKGLEWVSAI SGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 6) D5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVMKMSWVRQAPGKGLEWVSAI SGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDW GQGTLVTVSS(SEQ ID NO: 8) E5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSTGSMSWVRQAPGKGLEWVSAI SGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLY LFDW GQGTLVTVSS(SEQ ID NO: 10) C8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSLQTMSWVRQAPGKGLEWVSAISG SGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 12) F8 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAI SG SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 14) F8 VH V5LEVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAI SG SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 16) F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSQARMSWVRQAPGKGLEWVSAISG SGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 18) B7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFDMSWVRQAPGKGLEWVSAISG SGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FDY WGQGTLVTVSS(SEQ ID NO: 20) E8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDMHMSWVRQAPGKGLEWVSAI SGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FD YWGQGTLVTVSS(SEQ ID NO: 22) G9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSHMQMSWVRQAPGKGLEWVSAI SGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK STHLYL FD YWGQGTLVTVSS(SEQ ID NO: 24) F16 EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSAISG SGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK AHNAFDY W GQGTLVTVSR(SEQ ID NO: 26) 4A1-F16EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGASWVRQAPGKGLEWVSAI SG SGGSTYYADSVKGRFTISRIDNISKNTLYLQMNSLRAEDTAVYYCAK AHNAFDY W GQGTLVTVSR (SEQ ID NO: 28)

TABLE 3 Nucleotide sequences of the light chains (VL) of anti-ED-A andanti-tenascin C antibodies. VL CDR1 sequences are underlined; VLCDR2 sequences are in italics and underlined; VL CDR 3 sequencesare in bold and underlined. Antibody VL domain H1GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTCTGCGTGGTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 63) B2GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGTGGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 65) C5GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTTGCATTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 67) D5GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAATGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 69) E5GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTTGCGCATTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 71) C8GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTTCCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 73)F8 VL GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 75)F8 VL GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA K18RGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 77) F1GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCUCTCCTGCAGGGCCAGTCAGAGTGTTAGCGCGCCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 79) B7GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTGGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 81) E8GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTCGTCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 83) G9GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCACTGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTAT GGTGCA TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA TTACTGTCAGCAGATGCGTGGTCGGCCGCCG ACGTTCGGCCAAGGGACCAA GGTGGAAATCAAA (SEQ ID NO: 85)F16 and TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAG 4A1-F16TCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTAT GGTAAAAACAAC CGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACT GTAACTCCTCTGTTTATACTATGCCGCCCGTGGTA TTCGGCGGAGGGACCAA GCTGACCGTCCTA(SEQ ID NO: 87)

TABLE 4 Amino acid sequences of the light chains (VL) of anti-ED-A andanti-tenascin C antibodies. VL CDR1 sequences are underlined; VHCDR2 sequences are in italics and underlined; VH CDR 3 sequencesare in bold and underlined. Antibody VL domain H1EIVLTQSPGTLSLSPGEKATLSCRASQSVSSAWLAVVYQQKPGQAPRLLIY GASS RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 64) B2EIVLTQSPGTLSLSPGEKATLSCRASQSVSVAFLAINYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 66) C5EIVLTQSPGILSLSPGEKATLSCRASQSVSLHFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 68) D5EIVLTQSPGILSLSPGEKATLSCRASQSVSNAFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 70) E5EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAHLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ M RGRPP TFGQGTKVEIK (SEQ ID NO: 72)C8 EIVLTQSPGTLSLSPGEKATLSCRASQSVSLPFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 74)F8 VL EIVLTQSPGILSLSPGEKATLSCRASQSVSMPFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 76)F8 VL EIVLIQSPGILSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIY GASSR K18R ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 78) F1EIVLTQSPGTLSLSPGEKATLSCRASQSVSAPFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 80) B7EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 82) E8EIVLTQSPGTLSLSPGEKATLSCRASQSVSSSFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 84) G9EIVLTQSPGTLSLSPGEKATLSCRASQSVSTAFLAWYQQKPGQAPRLLIY GASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ MRGRPP TFGQGTKVEIK (SEQ ID NO: 86)F16 and SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIY GKNNRP 4A1 F16S GIPDRFSGSSSGNTASLTITGAQAEDEADYYC NSSVYTMPPVV FGGGTKLTVL(SEQ ID NO: 88)

Sequences Disclosed in Application

(H1 VH domain) SEQ ID NO: 1GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCGCGGAGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (H1 VH domain) SEQ ID NO: 2EVQLVESGGGLVQPGGSLRLSCAASGFTFSPRRMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B2 VH domain)SEQ ID NO: 3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGCGGCTAAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (B2 VH domain) SEQ ID NO: 4EVQLVESGGGLVQPGGSLRLSCAASGFTFSAAKMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C5 VH domain)SEQ ID NO: 5GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCGATTACTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (C5 VH domain) SEQ ID NO: 6EVQLVESGGGLVQPGGSLRLSCAASGFTFSPITMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (D5 VH domain)SEQ ID NO: 7GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGTGATGAAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (D5 VH domain) SEQ ID NO: 8EVQLVESGGGLVQPGGSLRLSCAASGFTFSVMKMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E5 VH domain)SEQ ID NO: 9GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCACTGGTTCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (E5 VH domain) SEQ ID NO: 10EVQLVESGGGLVQPGGSLRLSCAASGFTFSTGSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C8 VH domain)SEQ ID NO: 11GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTTCAGACTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (C8 VH domain) SEQ ID NO: 12EVQLVESGGGLVQPGGSLRLSCAASGFTFSLQTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F8 VH domain)SEQ ID NO: 13GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (F8 VH domain) SEQ ID NO: 14EVQLVESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F8 VH V5L domain)SEQ ID NO: 15GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (F8 VH V5L domain) SEQ ID NO: 16EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F1 VH domain)SEQ ID NO: 17GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTAGGCGCGTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (F1 VH domain) SEQ ID NO: 18EVQLVESGGGLVQPGGSLRLSCAASGFTFSQARMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B7 VH domain)SEQ ID NO: 19GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCATTTTGATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (B7 VH domain) SEQ ID NO: 20EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFDMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E8 VH domain)SEQ ID NO: 21GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGATATGCATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (E8 VH domain) SEQ ID NO: 22EVQLVESGGGLVQPGGSLRLSCAASGFTFSDMHMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (G9 VH domain)SEQ ID NO: 23GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCATATGCAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT (G9 VH domain) SEQ ID NO: 24EVQLVESGGGLVQPGGSLRLSCAASGFTFSHMQMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F16 VH domain)SEQ ID NO: 25GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGGTATGGTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCGCATAATGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCGAGA (F16 VH domain) SEQ ID NO: 26EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSR (4A1-F16 VH domain)SEQ ID NO: 27GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGGTATGGTGCGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCGCATAATGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCGAGA (4A1-F16 VH domain) SEQ ID NO: 28EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGASWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSR (H1 VH CDR1)SEQ ID NO: 29 CCGCGGAGG (H1 VH CDR1) SEQ ID NO: 30 PRR (B2 VH CDR1)SEQ ID NO: 31 GCGGCTAAG (B2 VH CDR1) SEQ ID NO: 32 AAK (C5 VH CDR1)SEQ ID NO: 33 CCGATTACT (C5 VH CDR1) SEQ ID NO: 34 PIT (D5 VH CDR1)SEQ ID NO: 35 GTGATGAAG (D5 VH CDR1) SEQ ID NO: 36 VMK (E5 VH CDR1)SEQ ID NO: 37 ACTGGTTCT (E5 VH CDR1) SEQ ID NO: 38 TGS (C8 VH CDR1)SEQ ID NO: 39 CTTCAGACT (C8 VH CDR1) SEQ ID NO: 40 LQT(F8 VH and F8 VH V5L CDR1) SEQ ID NO: 41 CTGTTTACG(F8 VH and F8 VH V5L CDR1) SEQ ID NO: 42 LFT (F1 VH CDR1) SEQ ID NO: 43TAGGCGCGT (F1 VH CDR1) SEQ ID NO: 44 QAR (B7 VH CDR1) SEQ ID NO: 45CATTTTGAT (B7 VH CDR1) SEQ ID NO: 46 HFD (E8 VH CDR1) SEQ ID NO: 47GATATGCAT (E8 VH CDR1) SEQ ID NO: 48 DMH (G9 VH CDR1) SEQ ID NO: 49CATATGCAG (G9 VH CDR1) SEQ ID NO: 50 HMQ (F16 VH CDR1) SEQ ID NO: 51CGGTATGGTATGAGC (F16 VH CDR1) SEQ ID NO: 52 RYGMS (4A1-F16 VH CDR1)SEQ ID NO: 53 CGGTATGGTGCGAGC (4A1-F16 VH CDR1) SEQ ID NO: 54 RYGAS(H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, E8 and G9 VH CDR2)SEQ ID NO: 55 AGTGGTAGTGGTGGTAGC(H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, E8 and G9 VH CDR2)SEQ ID NO: 56 SGSGGS (F16 and 4A1-F16 VH CDR2) SEQ ID NO: 57GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC(F16 and 4A1-F16 VH CDR2) SEQ ID NO: 58 AISGSGGSTYYADSVKG(H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, E8 and G9 VH CDR3)SEQ ID NO: 59 AGTACTCATTTGTATCTT(H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, 87, E8 and G9 VH CDR3)SEQ ID NO: 60 STHLYL (F16 and 4A1-F16 VH CDR3) SEQ ID NO: 61GCGCATAATGCTTTTGACTAC (F16 and 4A1-F16 VH CDR3) SEQ ID NO: 62 AHNAFDY(H1 VL domain) SEQ ID NO: 63GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTCTGCGTGGTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (H1 VL domain)SEQ ID NO: 64EIVLTQSPGTLSLSPGEKATLSCRASQSVSSAWLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B2 VL domain) SEQ ID NO: 65GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGGCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGTGGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (B2 VL domain)SEQ ID NO: 66EIVLTQSPGTLSLSPGEKATLSCRASQSVSVARAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C5 VL domain) SEQ ID NO: 67GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTTGCATTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (C5 VL domain)SEQ ID NO: 68EIVLTQSPGTLSLSPGEKATLSCRASQSVSLHFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (D5 VL domain) SEQ ID NO: 69GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAATGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (D5 VL domain)SEQ ID NO: 70EIVLIQSPGTLSLSPGEKATLSCRASQSVSNAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E5 VL domain) SEQ ID NO: 71GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTTGCGCATTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (E5 VL domain)SEQ ID NO: 72EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAHLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C8 VL domain) SEQ ID NO: 73GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTTCCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (C8 VL domain)SEQ ID NO: 74EIVLTQSPGTLSLSPGEKATLSCRASQSVSLPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F8 VL domain) SEQ ID NO: 75GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (F8 VL domain)SEQ ID NO: 76EIVLTQSPGTLSLSPGEKATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F8 VL K18R domain) SEQ ID NO: 77GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGIGTTAGCATGCCGTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATITTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (F8 VL K18R domain)SEQ ID NO: 78EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F1 VL domain) SEQ ID NO: 79GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGCGCCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (F1 VL domain)SEQ ID NO: 80EIVLTQSPGILSLSPGEKATLSCRASQSVSAPFLAWYQQKPGQAPRLLIVGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B7 VL domain) SEQ ID NO: 81GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTGGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (B7 VL domain)SEQ ID NO: 82EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E8 VL domain) SEQ ID NO: 83GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTCGTCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (E8 VL domain)SEQ ID NO: 84EIVLTQSPGTLSLSPGEKATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (G9 VL domain) SEQ ID NO: 85GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCACTGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (G9 VL domain)SEQ ID NO: 86EIVLTQSPGTLSLSPGEKATLSCRASQSVSTAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F16 and 4A1-F16 VL domain)SEQ ID NO: 87TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCTCTGTTTATACTATGCCGCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA(F16 and 4A1-F16 VL domain) SEQ ID NO: 88SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPWFGGGTKLTVL (H1 VL CDR1) SEQ ID NO: 89TCTGCGTGG (H1 VL CDR1) SEQ ID NO: 90 SAW (B2 VL CDR1) SEQ ID NO: 91GTGGCTTTT (B2 VL CDR1) SEQ ID NO: 92 VAF (C5 VL CDR1) SEQ ID NO: 93TTGCATTTT (C5 VL CDR1) SEQ ID NO: 94 LHF (D5 VL CDR1) SEQ ID NO: 95AATGCTTTT (D5 VL CDR1) SEQ ID NO: 96 NAF (E5 VL CDR1) SEQ ID NO: 97CTTGCGCAT  (E5 VL CDR1) SEQ ID NO: 98 LAH (C8 VL CDR1) SEQ ID NO: 99CTTCCTTTT (C8 VL CDR1) SEQ ID NO: 100 LPF (F8 VL and F8 VL K18R CDR1)SEQ ID NO: 101 ATGCCGTTT (F8 VL and F8 VL K18R CDR1) SEQ ID NO: 102 MPF(F1 VL CDR1) SEQ ID NO: 103 GCGCCITTT (F1 VL CDR1) SEQ ID NO: 104 APF(B7 VL CDR1) SEQ ID NO: 105 CTGGCTTTT (B7 VL CDR1) SEQ ID NO: 106 LAF(E8 VL CDR1) SEQ ID NO: 107 TCGTCTTTT (E8 VL CDR1) SEQ ID NO: 108 SSF(G9 VL CDR1) SEQ ID NO: 109 ACTGCTTTT  G9 VL CDR1) SEQ ID NO: 110 TAF(F16 and 4A1-F16 VL CDR1) SEQ ID NO: 111CAAGGAGACAGCCTCAGAAGCTATTATGCAAGC (F16 and 4A1-F16 VL CDR1)SEQ ID NO: 112 QGDSLRSYYAS(H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR2)SEQ ID NO: 113 GGTGCATCCAGCAGGGCCACT(H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR2)SEQ ID NO: 114 GASSRAT (F16 and 4A1-F16 VL CDR2) SEQ ID NO: 115GGTAAAAACAACCGGCCCTCA (F16 and 4A1-F16 VL CDR2) SEQ ID NO: 116 GKNNRPS(H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR3)SEQ ID NO: 117 ATGCGTGGTCGGCCGCCG(H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR3)SEQ ID NO: 118 MRGRPP (F16 and 4A1-F16 VL CDR3) SEQ ID NO: 119AACTCCTCTGTTTATACTATGCCGCCCGTGGTA (F16 and 4A1-F16 VL CDR3)SEQ ID NO: 120 NSSVYTMPPVV (F8, F16 and 4A1-F16 linker) SEQ ID NO: 121GGCGGTAGCGGAGGG (F8, F16 and 4A1-F16 linker) SEQ ID NO: 122 GGSGG(H1 VH V5L domain) SEQ ID NO: 123EVQLLESGGGLVQPGGSLRLSCAASGFTFSPRRMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFT1SRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B2 VH V5L domain)SEQ ID NO: 124EVQLLESGGGLVQPGGSLRLSCAASGFTFSAAKMSAWRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C5 VH V5L domain)SEQ ID NO: 125EVQLLESGGGLVQPGGSLRLSCAASGFTFSPITMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (D5 VH V5L domain)SEQ ID NO: 126EVQLLESGGGLVQPGGSLRLSCAASGFTFSVMKMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E5 VH V5L domain)SEQ ID NO: 127EVQLLESGGGLVQPGGSLRLSCAASGFTFSTGSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C8 VH V5L domain)SEQ ID NO: 128EVQLLESGGGLVQPGGSLRLSCAASGFTFSLQTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F1 VH V5L domain)SEQ ID NO: 129EVQLLESGGGLVQPGGSLRLSCAASGFTFSQARMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B7 VH V5L domain)SEQ ID NO: 130EVQLLESGGGLVQPGGSLRLSCAASGFTFSHFDMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E8 VH V5L domain)SEQ ID NO: 131EVQLLESGGGLVQPGGSLRLSCAASGFTFSDMHMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (G9 VH V5L domain)SEQ ID NO: 132EVQLLESGGGLVQPGGSLRLSCAASGFTFSHMQMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (H1 VL K18R domain)SEQ ID NO: 133EIVLTQSPGTLSLSPGERATLSCRASQSVSSAWLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B2 VL K18R domain) SEQ ID NO: 134EIVLTQSPGTLSLSPGERATLSCRASQSVSVAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C5 VL K18R domain) SEQ ID NO: 135EIVLTQSPGTLSLSPGERATLSCRASQSVSLHFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (D5 VL K18R domain) SEQ ID NO: 136EIVLTQSPGTLSLSPGERATLSCRASQSVSNAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E5 VL K18R domain) SEQ ID NO: 137EIVLTQSPGTLSLSPGERATLSCRASQSVSLAHLAWYQQKPGQAPRLLIVGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C8 VL K18R domain) SEQ ID NO: 138EIVLTQSPGTLSLSPGERATLSCRASQSVSLPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F1 VL K18R domain) SEQ ID NO: 139EIVLTQSPGTLSLSPGERATLSCRASQSVSAPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B7 VL K18R domain) SEQ ID NO: 140EIVLTQSPGTLSLSPGERATLSCRASQSVSLAFLAWYQQKPGQAPRLLIVGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E8 VL K18R domain) SEQ ID NO: 141EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (G9 VL K18R domain) SEQ ID NO: 142EIVLTQSPGTLSLSPGERATLSCRASQSVSTAFLAWYQQKPGQAPRLLIVGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK

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What is claimed is:
 1. A method of delivering a molecule to theneovasculature of endometriotic tissue in a human or animal, wherein themolecule is conjugated to a specific binding member which binds the ED-Aisoform of fibronectin to form a conjugate and the method comprisesadministering the conjugate to the human or animal.
 2. The method ofclaim 1, wherein the molecule is a detectable label.
 3. The method ofclaim 1, wherein the molecule is a radioisotope.
 4. The specific bindingmember of claim 1, wherein the molecule is a bioactive molecule selectedfrom the group consisting of a cytokine, hormone, a therapeuticradioisotope and a cytotoxic drug, and wherein the bioactive molecule isoptionally conjugated to the specific binding member by a cleavablelinker.
 5. The method of claim 4, wherein the specific binding member isconjugated to the bioactive molecule by a cleavable linker.
 6. Themethod of claim 1, wherein the specific binding member is an anti-EDAantibody comprising a VH domain comprising a framework and a set ofcomplementarity determining regions HCDR1, HCDR2 and HCDR3, whereinHCDR1 has amino acid sequence SEQ ID NO: 42, HCDR2 has amino acidsequence SEQ ID NO: 56, HCDR3 has amino acid sequence SEQ ID NO: 60; anda VL domain comprising a set of complementarity determining regionsLCDR1, LCDR2 and LCDR3 and a framework wherein LCDR1 has amino acidsequence SEQ ID NO: 102, LCDR2 has amino acid sequence SEQ ID NO: 114,and LCDR3 has amino acid sequence SEQ ID NO:
 118. 7. The method of claim6, wherein the VH domain framework is a human germline framework, andwherein the VH domain optionally has amino acid sequence SEQ ID NO: 16.8. The method of claim 7, wherein the VH domain has amino acid sequenceSEQ ID NO:
 16. 9. The method of claim 6, wherein the VL domain frameworkis a human germline framework, wherein the VL domain optionally hasamino acid sequence SEQ ID NO:
 78. 10. The method of claim 9, whereinthe VL domain has amino acid sequence SEQ ID NO:
 78. 11. The method ofclaim 6, wherein the specific binding member comprises a single chainFv.
 12. The method of claim 11, wherein the binding member is a smallimmunoprotein (SIP).
 13. The method of claim 6, wherein the specificbinding member is a diabody.